Compounds for targeted immunotherapy

ABSTRACT

Compounds for targeted immunotherapy, compositions comprising the compounds and use of the compounds in the treatment of diseases such as cancer are disclosed. The compounds having the structure of formula TM-Ln-AM, wherein TM is a targeting moiety, AM is an activating moiety that is capable of activating a human dendritic cell, NK cell, or tumor cell, or a combination thereof, Ln is a linker, and n is an integer selected from 0 and 1.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of, and priority to, Chinese PatentApplication Serial No. 201210248481.9, filed on Jul. 18, 2012, theentire disclosure of which is hereby incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to compounds for targeted immunotherapy,as well as compositions comprising the same. Further, the presentinvention relates to the use of the compounds in the treatment ofdiseases such as cancer.

BACKGROUND OF THE INVENTION

Therapeutic antibodies have been used in clinical applications for overtwenty years. Currently, there are fifteen anti-tumor antibody drugs inclinic, including Rituxan (1997), Herceptin (1998), Mylotarg (2000),Campath (2001), Zevalin (2002), Bexxer (2003), Avastin (2004), Erbitux(2004), Vectibix (2006); Arzerra (2009); Benlysta (2011); Yervoy (2011);Adcetris (2011); Perjeta (2012); and Kadcyla (2013). These antibodiestarget mainly four molecules: EGFR, Her2, CD20 and VEGF.

In general, therapeutic antibodies kill tumor cells via three mechanisms(Scott A M, Wolchok J D, Old L J. Antibody therapy of cancer. Nat RevCancer. (2012), 12:278-87): (1) Direct antibody action, that is,blockade or agonist activity of ligand/receptor signaling, induction ofapoptosis, and delivery of drugs or cytotoxic agents. Antibody receptoractivation activity can produce direct tumor cell killing effect. Forexample, some antibodies can bind to receptors on the surface of tumorcells, activate the receptor, leading to apoptosis (e.g., inmitochondria). Antibodies can also mediate tumor cell killing byreceptor-antagonistic activity. For example, certain antibodies can bindto cell surface receptors and block dimerization, kinase activation anddownstream signaling, thereby inhibiting proliferation and promoteapoptosis. Binding of antibodies to an enzyme can lead toneutralization, signal abrogation, and cell death. (2) Throughimmune-mediated cell killing mechanisms include complement-dependentcytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity(ADCC), T cell function regulation, etc. Immune-mediated killing oftumor cells can be accomplished through the following ways: induction ofphagocytosis, complement activation, antibody-dependent cell-mediatedcytotoxicity, genetically modified T cells being targeted to the tumorby single-chain variable fragment (scFv), through antibody-mediatedantigenic cross presentation to dendritic cell to activate T cells,inhibition of T cell inhibitory receptors, such as cytotoxic Tlymphocyte-associated antigen 4 (CTLA4). Of them, the Fc portion of theantibody feature is especially important for CDC and ADCC-mediated tumorcell killing effect. (3) Specific effect of antibody on tumorvasculature and matrix, through trapping of vascular receptor antagonistor ligand to induce vascular and stromal cells ablation, including:stromal cell inhibition, delivery of toxins to stromal cells, anddelivery of toxins to the vasculature. (Scott A M, Wolchok J D, Old L J.Antibody therapy of cancer. Nat Rev Cancer. 2012, 12 (4):278-87).

Therapeutic monoclonal antibody drugs have advanced anti-cancer drugresearch and development. However, some issues still need further studyto be solved, such as antibody immunogenicity, tolerance of long-termuse of tumor target, and long-term effects of simple single blockade ofsignal transduction pathway. In short, a simple majority of antibodiesare difficult to achieve long-term efficient inhibition and killing oftumor cells.

In 1964, “Nature” magazine presented the new idea of antibody-drugconjugates (ADC) technology, which in recent years have seenbreakthroughs. ADC covalently links antibody with a highly toxic drug(toxin) through a chemical linker (linker). Antibody recognizes cancercell surface antigen molecule, the endocytosis ADC brings it intocytoplasm, and in particular intracellular environment toxins releasedafter hydrolysis of the linker kills cells.

Seattle Genetics has developed such drug Brentuximab Vedotin (trade nameAdcetris) that has been approved by the FDA to market. It is monomethylauristatin E (MMAE), a synthetic toxic anti-cancer drug, coupled withantibody targeting lymphoma cells specific CD30 molecule, with improvedefficacy of killing tumor cells.

Currently, there are dozens of such ADC drugs in clinical trials. Amongthem, Genentech and Immunogen jointly developed trastuzumab coupled withmaytansines as a drug named ado-trastuzumab emtansine (Kadcyla), alsoknown as T-DM1, to treat breast cancer. In February 2013, the FDA hasapproved T-DM1 for human epidermal growth factor receptor 2(Her2)-positive metastatic breast cancer. Maytansines is a smallmolecule toxin that can bind tubulin and prevent formation ofmicrotubules by forming non-reducing dual-maleimide-propanediol complex.Trastuzumab acts on breast cancer and gastric cancer by targeting humanHer2. It was approved for Her2-positive cancer. However, trastuzumabcannot promote apoptosis of all of the Her2 positive cells. T-DM1combines the selective targeting Her2 receptor trastuzumab with thepotent cytotoxic agent maytansine to kill tumor cells. T-DM1 antibodybinds Her2 receptors, causing cellular internalization of themaytansines released from conjugates, thereby killing the tumor cells.T-DM1 has better overall efficacy and pharmacokinetic properties and lowtoxicity.

Traditional small molecule chemotherapeutic drugs have strong toxicityand pharmacokinetic advantages, but in the process of treatment oftumors may affect other physiological targets with serious side effects.Antibody-drug conjugates combines targeting function and small moleculedrug with particular pharmacokinetics. The structure of antibody-drugconjugates is the attachment of a monoclonal antibody with targetingfunction to a compound with specific pharmacological properties. Thistechnique requires the therapeutic antibody have binding specificity toa target, to be coupled to a molecule with therapeutic effect or otherfunctions such as cyto-toxins. Many factors affect the effect of thistype of antibodies, such as endocytosis of the coupled antibody,stability of the coupling, and release and killing activity of thetoxins.

Toxin molecules currently being used include tubulin inhibitorsAuristatin analogues monomethyl auristatin E, monomethyl auristatin Fand maytansine. Monomethyl auristatin E is a synthetic microtubulepolymer inhibitor that can inhibit microtubule aggregation, interferetumor cell mitosis and induce apoptosis (Naumovski L and Junutula J R.Glembatumumab vedotin, a conjugate of an anti-glycoproteinnon-metastatic melanoma protein B mAb and monomethyl auristatin E fortreatment of melanoma and breast cancer. Curr Opin Mol Ther 2003; 12(2): 248-57. Francisco J A, Cerveny C G et al. cAC10-vcMMAE, ananti-CD30-monomethyl auristatin E conjugate with potent and selectiveantitumor activity. Blood 102 (4): 1458-65. Monomethyl auristatin F isan anti-mitotic Auristatin derivative with a charged phenylalanineresidue at C terminus. In comparison to uncharged MMAE it minimizesdamage to cell signaling pathway and minimizes cytotoxicity. A largenumber of test with CD30 cells found thatmAb-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl-MMAF(mAb-L1-MMAF) has a toxicity that is 2,200 times stronger than MMAF only(Doronina S O et al., Enhanced activity of monomethylauristatin Fthrough monoclonal antibody delivery: effects of linker technology onefficacy and toxicity. Bioconjug Chem, 2006; 17 (1): p 114-24).Maytansine is an antimitotic agent acting as an inhibitor of tubulinpolymerization, thus interfering with formation of microtubules in thecell nucleus. Maytansine also inhibits DNA, RNA, and protein synthesis,with the greatest effect being seen on DNA synthesis.

Antibodies-drug conjugates have direct and indirect anti-cancer effect.The antibody blocks or activates ligand/receptor signaling, inducesapoptosis, and at the same time can present or deliver payload drugdirectly or indirectly (such as a drug, toxin, small interfering RNA orradioisotope) to the tumor cells. Therapeutic antibody drug conjugateutilizes dual characteristics of the antibody and the coupled drug,first is the binding function that it specifically binds to the targetmolecule, second is the tumor cell killing function of the antibodyitself, and the third is the particular effect of the conjugated drug.Current antibody-drug conjugates drugs are limited in how to kill tumorcells directly. However, because of the tough requirement oftechnologies in antibody, linker molecule, toxin molecules, andconjugation, as well as the limitation of bringing toxins within thetumor microenvironment molecules, there are still some difficulties inactual clinical studies.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a compound having thestructure of Formula (I):

TM-Ln-AM  (I),

wherein TM is a targeting moiety, AM is an activating moiety that iscapable of activating human immune cells, including but not limited todendritic cells, macrophages, monocytes, myeloid-derived suppressorcells, NK cells, B cells, T cells or tumor cells, or a combinationthereof, Ln is a linker, and n is an integer selected from 0 and 1.

In some embodiments, the human dendritic cell is a plasmacytoiddendritic cell. In some embodiments, the human dendritic cell is amyeloid dendritic cell.

In some embodiments, the AM is capable of binding specifically to humantoll like receptor 7 (TLR7) and/or TLR8; or activating human immunecells via TLR7 and/or TLR8.

In another aspect, the present invention provides a compound having thestructure of Formula (I):

TM-Ln-AM  (I),

wherein TM is a targeting moiety, AM is an activating moiety that iscapable of binding specifically to human TLR7 and/or TLR8, or activatinghuman immune cells via TLR7 and/or TLR8, Ln is a linker, and n is aninteger selected from 0 and 1.

In some embodiments, wherein the AM is capable of binding specificallyto both human TLR7 and TLR8; or activating human immune cells via TLR7and/or TLR8.

In some embodiments, wherein when TM comprises an antibody, AM does notcomprise a CpG oligonucleotide.

In some embodiments, wherein the targeting moiety is capable of bindingto a tumor cell specifically or preferably in comparison to a non-tumorcell. In some embodiments, the tumor cell is of a carcinoma, a sarcoma,a lymphoma, a myeloma, or a central nervous system cancer.

In some embodiments, the targeting moiety is capable of binding to atumor antigen specifically or preferably in comparison to a non-tumorantigen. In some embodiments, the tumor antigen is selected from thegroup consisting of: CD2, CD19, CD20, CD22, CD27, CD33, CD37, CD38,CD40, CD44, CD47, CD52, CD56, CD70, CD79, and CD137. In someembodiments, the tumor antigen is selected from the group consisting of:4-1BB, 5T4, AGS-5, AGS-16, Angiopoietin 2, B7.1, B7.2, B7DC, B7H1, B7H2,B7H3, BT-062, BTLA, CAIX, Carcinoembryonic antigen, CTLA4, Cripto, ED-B,ErbB1, ErbB2, ErbB3, ErbB4, EGFL7, EpCAM, EphA2, EphA3, EphB2, FAP,Fibronectin, Folate Receptor, Ganglioside GM3, GD2,glucocorticoid-induced tumor necrosis factor receptor (GITR), gp100,gpA33, GPNMB, ICOS, IGF1R, Integrin αν, Integrin ανβ, KIR, LAG-3, LewisY, Mesothelin, c-MET, MN Carbonic anhydrase IX, MUC1, MUC16, Nectin-4,NKGD2, NOTCH, OX40, OX40L, PD-1, PDL1, PSCA, PSMA, RANKL, ROR1, ROR2,SLC44A4, Syndecan-1, TACI, TAG-72, Tenascin, TIM3, TRAILR1, TRAILR2,VEGFR-1, VEGFR-2, VEGFR-3, and variants thereof.

In some embodiments, the targeting moiety comprises an immunoglobulin, aprotein, a peptide, a small molecule, a nanoparticle, or a nucleic acid.

In some embodiments, the targeting moiety comprises an antibody, or afunctional fragment thereof. In some embodiments, the antibody isselected from the group consisting of: Rituxan (rituximab), Herceptin(trastuzumab), Erbitux (cetuximab), Vectibix (Panitumumab), Arzerra(Ofatumumab), Benlysta (belimumab), Yervoy (ipilimumab), Perjeta(Pertuzumab), Tremelimumab, Nivolumab, Dacetuzumab, Urelumab, MPDL3280A,Lambrolizumab, and Blinatumomab.

In some embodiments, the targeting moiety comprises a Fab, Fab′,F(ab′)2, single domain antibody, T and Abs dimer, Fv, scFv, dsFv,ds-scFv, Fd, linear antibody, minibody, diabody, bispecific antibodyfragment, bibody, tribody, sc-diabody, kappa (lamda) body, BiTE, DVD-Ig,SIP, SMIP, DART, or an antibody analogue comprising one or more CDRs.

In some embodiments, the targeting moiety comprises a ATWLPPRpolypeptide of VEGFR, Thrombospondin-1 mimetics, CDCRGDCFCG (cyclic)polypeptide, SCH 221153 fragment, NCNGRC (cyclic) polypeptide,CTTHWGFTLC polypeptide, CGNKRTRGC polypeptide (LyP-1), Octreotide,Vapreotide, Lanreotide, C-3940 polypeptide, Decapeptyl, Lupron, Zoladex,or Cetrorelix.

In some embodiments, the targeting moiety comprises folic acid or aderivative thereof.

In some embodiments, the targeting moiety comprises extracellulardomains (ECD) or soluble form of PD-1, CTLA4, BTLA, KIR, TIM3, 4-1BB,LAG3, full length of partial of a surface ligand amphiregulin,betacellulin, EGF, ephrin, epigen, epiregulin, IGF, neuregulin, TGF,TRAIL, or VEGF.

In some embodiments, the targeting moiety comprises a nanoparticle.

In some embodiments, the targeting moiety comprises an aptamer.

In some embodiments, the activating moiety comprises: (a)single-stranded RNA (ssRNA), preferably ORN02, ORN06, ssPoly(U),ssRNA40, ssRNA41, ssRNA-DR, or Poly(dT); or (b) a toll like receptorligand (TLRL) analogs, preferably CL075, CL097, CL264, CL307,Gardiquimod, Loxoribine, Imiquimod, or Resiquimod.

In some embodiments, the linker is selected from the group consisting ofa hydrazine group, a polypeptide, a disulfide group, and a thioethergroup.

In some embodiments, the linker is enzymatic cleavable. In someembodiments, the linker is not enzymatic cleavable.

In some embodiments, the present invention provides a pharmaceuticalcomposition comprising a compound as provided herein, or apharmaceutically acceptable salt thereof, and one or morepharmaceutically acceptable carriers.

In some embodiments, the pharmaceutical composition further comprises asecond chemotherapeutic agent. In some embodiments, the chemotherapeuticagent is selected from the group consisting of tamoxifen, raloxifene,anastrozole, exemestane, letrozole, imatanib, paclitaxel,cyclophosphamide, lovastatin, minosine, gemcitabine, cytarabine,5-fluorouracil, methotrexate, docetaxel, goserelin, vincristine,vinblastine, nocodazole, teniposide etoposide, gemcitabine, epothilone,vinorelbine, camptothecin, daunorubicin, actinomycin D, mitoxantrone,acridine, doxorubicin, epirubicin, or idarubicin.

In yet another aspect, the present invention provides a method fortreating a disease condition in a subject that is in need of suchtreatment, comprising: administering to the subject a pharmaceuticalcomposition comprising a therapeutically effective amount of thecompound provided herein or a pharmaceutically acceptable salt thereof,and a pharmaceutical acceptable carrier.

In some embodiments, the diseases condition is tumor. In someembodiments, the disease condition comprises abnormal cellproliferation. In some embodiments, the abnormal cell proliferationcomprises a pre-cancerous lesion. In some embodiments, the abnormalproliferation is of cancer cells.

In some embodiments, the cancer is selected from the group consistingof: breast cancer, colorectal cancer, diffuse large B-cell lymphoma,endometrial cancer, follicular lymphoma, gastric cancer, glioblastoma,head and neck cancer, hepatocellular cancer, lung cancer, melanoma,multiple myeloma, ovarian cancer, pancreatic cancer, prostate cancer,and renal cell carcinoma.

In a further aspect, the present invention provides a compound havingthe structure of Formula (I):

TM-Ln-AM  (I),

wherein TM is a targeting moiety, AM is an activating moiety, Ln is alinker, and n is an integer selected from 0 and 1,with the proviso that when TM comprises an antibody, AM does notcomprise a CpG oligonucleotide or a therapeutic agent.

In some embodiments, the activating moiety comprises a TLR ligand, aNod-Like Receptor ligand, a RIG-Like Receptor ligand, a CLR ligand, aCDS ligand, or a inflammasome inducers.

In some embodiments, the activating moiety comprises a ligand of one ormore TLRs (toll like receptor ligand, TLRL) selected from the groupconsisting of: TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, TLR7/TLR8, TLR9, andTLR10.

In some embodiments, the activating moiety comprises:

(i) a ligand of TLR2 selected from the group consisting of: (a) a heatkilled bacteria product, preferably HKAL, HKEB, HKHP, HKLM, HKLP, HKLR,HKMF, HKPA, HKPG, or HKSA, HKSP, and (b) a cell-wall components product,preferably LAM, LM, LPS, LTA, LTA, PGN, FSL, Pam2CSK4, Pam3CSK4, orZymosan;

(ii) a ligand of TLR3 selected from the group consisting of: Poly (I:C)and Poly (A:U);

(iii) a ligand of TLR4 selected from the group consisting of: LPS andMPLA;

(iv) a ligand of TLR5 selected from the group consisting of: FLA andFlagellin;

(v) a ligand of TLR7/8 selected from the group consisting of: ORN02,ORN06, ssPoly(U), ssRNA40, ssRNA41, ssRNA-DR, Poly(dT), CL075, CL097,CL264, CL307, Gardiquimod, Loxoribine, Imiquimod, and Resiquimod;

(vi) a ligand of TLR9 selected from the group consisting of: ODN1585,ODN1668, ODN1826, ODN2006, ODN2007, ODN2216, ODN2336, ODN2395, and ODNM362;

(vii) a ligand of TLR10;

(viii) a ligand of nucleotide-oligomerization domain (NOD)-like selectedfrom the group consisting of: NOD1 agonist (C12-iE-DAP, iE-DAP,Tri-DAP), NOD2 agonist (L18-MDP, MDP, M-TriLYS, M-TriLYS-D-ASN,Murabutide, N-Glycolyl-MDP), and NOD1/NOD2 agonists (M-TriDAP, PGN);

(ix) a ligand of one or more RIG-I-Like receptors (RLR) selected fromthe group consisting of: 5′ppp-dsRNA, Poly (dA:dT), Poly(dG:dC), andPoly (I:C);

(x) a ligand of one or more C-type lectin receptors (CLR) selected fromthe group consisting of: Curdlan AL, HKCA, HKSC, WGP, Zymosan, andTrehalose-6,6-dibehenate;

(xi) a ligand of one or more Cytosolic DNA Sensors (CDS) selected fromthe group consisting of: c-GMP, c-G-AMP, c-G-GMP, c-A-AMP, c-di-AMP,c-di-IMP, c-di-GMP, c-di-UMP, HSV-60, ISD, pCpG, Poly (dA:dT),Poly(dG:dC), Poly (dA), and VACV-70; or

(xii) an inflammasome inducer selected from the group consisting of: (a)NLRP3 inflammasome protein complex, preferably alum Crystals, ATP, CPPDCrystals, Hermozoin, MSU Crystals, Nano-SiO2, Nigericin, and (b) AIM2inflammasome protein complex, preferably Poly (dA:dT).

In some embodiments, the activating moiety comprises IMO2134, IMO205,MGN-1703, MGN-1704, Agatolimod, SD-101, QAX-935, DIMS0150, PollinexQuattro, OM-174, Eritoran, TAK-242, NI-0101, Type I interferon, type IIinterferon, type III interferon, IL-12, IL-23, IL-18, IL-7 or IL-15.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 depicts conjugated compound characteristics. Binding ofTrastuzumab MC-vc-resiquimod and Trastuzumab-resiquimod conjugates toHer2 expressing L cells is compared with unconjugated Trastuzumab (as areference), control human IgG, and irrelevant human IgG1 antibody, asdetermined by FACS analysis.

FIG. 2 depicts in vitro antibody-dependent cellular cytotoxicity (ADCC)analysis of Trastuzumab, Trastuzumab MC-vc-TLRL and Trastuzumab MC-TLRLconjugates. PBMCs (effector cells) were added with SKBR3 cells (targetcells) into 96-well plates (ratio targets/effectors 1/60) for 17 hcontaining control human IgG1, trastuzumab, or Trastuzumab MC-vc-TLRLand Trastuzumab MC-TLRL conjugates at different concentrations. Allexperiments were performed in duplicate. Analysis was based on thenegative SKBR3 population determination. The dead cells were stained byLDH.

FIG. 3A depicts the percentages of DCs before and post enrichment. Thenumbers in upper two plots represent the percentages of DCs(HLA-DR+Lin−) of total cells before and after lineage depletion. Thenumbers in lower plots represent percentages of mDC (CD11C+CD123−) andpDC (CD123+CD11C−) of total DCs before and after lineage depletion.FIGS. 3B-3D depict analysis of cytokine production by purified humanDCs. Purified human DCs were plated in a 96-well plate and cultured withallogeneic untreated (medium) or treated different concentration ofTLRL, or Trastuzumab MC-vc-TLRL and Trastuzumab MC-TLRL directly for20-22 h in 37′C incubator. In a separate experiment, CetuximabMC-vc-TLRL and Cetuximab MC-TLRL in an increasing concentration weregiven to treat with human DCs. The supernatant were collected and humanIFN-α, IL-6, IL-12(p70) and TNF-α were analyzed by ELISA. Data are givenas mean±SD of triplicate cultures and are representative of independentexperiments from two of three healthy donors.

FIGS. 4A-4C depict therapeutic efficacy of Trastuzumab MC-vc-TLRL andTrastuzumab MC-TLRL in PDX gastric tumor model. BALB/c nu/nu nude mice6-8 week-old female mice bearing subcutaneous patient derived gastrictumor were treated intravenously with 10 mg/kg of TrastuzumabMC-vc-TLRL, Trastuzumab MC-TLRL, or unconjugated Trastuzumab or saline(12 mice per group). Treatment was performed weekly for a period of 45days. Therapy was initiated when tumors reached a size on average of 170mm³. FIG. 4A: Data represents mean tumor volumes (mean±SD). Tumor growthcurves were stopped when tumors reached a size of 2000 mm³. FIG. 4B:Body weight variations of the mice during and after therapy. Nodetectable weight loss was observed. FIG. 4C: Survival curves oftreatment and control groups; substantial prolongation for mice whichreceived Trastuzumab MC-vc-TLRL, or unconjugated Trastuzumab.

FIGS. 5A-5B depict therapeutic efficacy of Cetuximab MC-vc-TLRL in anude/H1650 human lung cancer model. BALB/c nu/nu nude mice 6-8 week-oldfemale mice bearing subcutaneous H1650 lung cancer cells were treatedintravenously with 10 mg/kg of Cetuximab MC-vc-TLRL, or unconjugatedCetuximab or saline (12 mice per group). Treatment was performed weeklyfor a period of 45 days. Therapy was initiated when tumors reached asize on average of 170 mm³. Data represents mean tumor volumes(mean±SD). Tumor growth curves were stopped when tumors reached a sizeof 2000 mm³. FIG. 5A: Therapeutic efficacy of Cetuximab MC-vc-TLRL inhuman lung cancer model. FIG. 5B: Body weight variations of the miceduring and after therapy. No detectable weight loss was observed.

DETAILED DESCRIPTION OF THE INVENTION

Several aspects of the invention are described below with reference toexample applications for illustration. It should be understood thatnumerous specific details, relationships, and methods are set forth toprovide a full understanding of the invention. One having ordinary skillin the relevant art, however, will readily recognize that the inventioncan be practiced without one or more of the specific details or withother methods. The present invention is not limited by the illustratedordering of acts or events, as some acts may occur in different ordersand/or concurrently with other acts or events.

Furthermore, not all illustrated acts or events are required toimplement a methodology in accordance with the present invention.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Furthermore, to the extent that the terms “including”,“includes”, “having”, “has”, “with”, or variants thereof are used ineither the detailed description and/or the claims, such terms areintended to be inclusive in a manner similar to the term “comprising”.

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviation,per the practice in the art. Alternatively, “about” can mean a range ofup to 20%, preferably up to 10%, more preferably up to 5%, and morepreferably still up to 1% of a given value. Alternatively, particularlywith respect to biological systems or processes, the term can meanwithin an order of magnitude, preferably within 5-fold, and morepreferably within 2-fold, of a value. Where particular values aredescribed in the application and claims, unless otherwise stated theterm “about” meaning within an acceptable error range for the particularvalue should be assumed.

I. DEFINITIONS AND ABBREVIATIONS

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in cellculture, molecular genetics, organic chemistry and nucleic acidchemistry and hybridization are those well-known and commonly employedin the art. Standard techniques are used for nucleic acid and peptidesynthesis. The techniques and procedures are generally performedaccording to conventional methods in the art and various generalreferences, which are provided throughout this document. Thenomenclature used herein and the laboratory procedures in analyticalchemistry, and organic synthetic described below are those well-knownand commonly employed in the art. Standard techniques, or modificationsthereof, are used for chemical syntheses and chemical analyses.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include di- and multivalentradicals, having the number of carbon atoms designated (i.e. C₁-C₁₀means one to ten carbons). Examples of saturated hydrocarbon radicalsinclude, but are not limited to, groups such as methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Anunsaturated alkyl group is one having one or more double bonds or triplebonds. Examples of unsaturated alkyl groups include, but are not limitedto, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. The term “alkyl,” unlessotherwise noted, is also meant to include those derivatives of alkyldefined in more detail below, such as “heteroalkyl.” Alkyl groups, whichare limited to hydrocarbon groups, are termed “homoalkyl”.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkane, as exemplified, but notlimited, by —CH₂CH₂CH₂CH₂—, and further includes those groups describedbelow as “heteroalkylene.” Typically, an alkyl (or alkylene) group willhave from 1 to 24 carbon atoms, with those groups having 10 or fewercarbon atoms being preferred in the present invention. A “lower alkyl”or “lower alkylene” is a shorter chain alkyl or alkylene group,generally having eight or fewer carbon atoms.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and at least one heteroatom selected fromthe group consisting of O, N, Si and S, and wherein the nitrogen andsulfur atoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) O, N and S and Si may beplaced at any interior position of the heteroalkyl group or at theposition at which the alkyl group is attached to the remainder of themolecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,and —CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may be consecutive, suchas, for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. Similarly, the term“heteroalkylene” by itself or as part of another substituent means adivalent radical derived from heteroalkyl, as exemplified, but notlimited by, —CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. Forheteroalkylene groups, heteroatoms can also occupy either or both of thechain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino,alkylenediamino, and the like). Still further, for alkylene andheteroalkylene linking groups, no orientation of the linking group isimplied by the direction in which the formula of the linking group iswritten. For example, the formula —C(O)₂R′— represents both —C(O)₂R′—and —R′C(O)₂—.

In general, an “acyl substituent” is also selected from the group setforth above. As used herein, the term “acyl substituent” refers togroups attached to, and fulfilling the valence of a carbonyl carbon thatis either directly or indirectly attached to the polycyclic nucleus ofthe compounds of the present invention.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” is mean to include, but not be limited to,trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, andthe like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent, which can be a single ring ormultiple rings (preferably from 1 to 3 rings), which are fused togetheror linked covalently. The term “heteroaryl” refers to aryl groups (orrings) that contain from one to four heteroatoms selected from N, O, andS, wherein the nitrogen and sulfur atoms are optionally oxidized, andthe nitrogen atom(s) are optionally quaternized. A heteroaryl group canbe attached to the remainder of the molecule through a heteroatom.Non-limiting examples of aryl and heteroaryl groups include phenyl,1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl,3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl,4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of the above notedaryl and heteroaryl ring systems are selected from the group ofacceptable substituents described below.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxyl)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) include both substituted and unsubstituted forms of theindicated radical. Preferred substituents for each type of radical areprovided below.

Substituents for the alkyl, and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) are generally referred to as “alkyl substituents”and “heteroakyl substituents,” respectively, and they can be one or moreof a variety of groups selected from, but not limited to: —OR′, ═O,═NR′, ═N—OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′,—CO₂R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′,—NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″,—NRSO₂R′, —CN and —NO₂ in a number ranging from zero to (2m′+1), wherem′ is the total number of carbon atoms in such radical. R′, R″, R′″ andR″″ each preferably independently refer to hydrogen, substituted orunsubstituted heteroalkyl, substituted or unsubstituted aryl, e.g., arylsubstituted with 1-3 halogens, substituted or unsubstituted alkyl,alkoxy or thioalkoxy groups, or arylalkyl groups. When a compound of theinvention includes more than one R group, for example, each of the Rgroups is independently selected as are each R′, R″, R′″ and R″″ groupswhen more than one of these groups is present. When R′ and R″ areattached to the same nitrogen atom, they can be combined with thenitrogen atom to form a 5-, 6-, or 7-membered ring. For example, —NR′R″is meant to include, but not be limited to, 1-pyrrolidinyl and4-morpholinyl. From the above discussion of substituents, one of skillin the art will understand that the term “alkyl” is meant to includegroups including carbon atoms bound to groups other than hydrogengroups, such as haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g.,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Similar to the substituents described for the alkyl radical, the arylsubstituents and heteroaryl substituents are generally referred to as“aryl substituents” and “heteroaryl substituents,” respectively and arevaried and selected from, for example: halogen, —OR′, ═O, ═NR′, ═N—OR′,—NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″,—OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl,in a number ranging from zero to the total number of open valences onthe aromatic ring system; and where R′, R″, R′″ and R″″ are preferablyindependently selected from hydrogen, (C₁-C₈)alkyl and heteroalkyl,unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C₁-C₄)alkyl,and (unsubstituted aryl)oxy-(C₁-C₄)alkyl. When a compound of theinvention includes more than one R group, for example, each of the Rgroups is independently selected as are each R′, R″, R′″ and R″″ groupswhen more than one of these groups is present.

Two of the aryl substituents on adjacent atoms of the aryl or heteroarylring may optionally be replaced with a substituent of the formula-T-C(O)—(CRR′)_(q)—U—, wherein T and U are independently —NR—, —O—,—CRR′— or a single bond, and q is an integer of from 0 to 3.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula -A-(CH₂)_(r)—B—, wherein A and B are independently —CRR′—, —O—,—NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or a single bond, and r is aninteger of from 1 to 4. One of the single bonds of the new ring soformed may optionally be replaced with a double bond. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula—(CRR′)_(s)—X—(CR″R′″)_(d)—, where s and d are independently integers offrom 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—.The substituents R, R′, R″ and R′″ are preferably independently selectedfrom hydrogen or substituted or unsubstituted (C₁-C₆) akyl.

As used herein, the term “heteroatom” includes oxygen (O), nitrogen (N),sulfur (S), phosphorus (P) and silicon (Si).

II. THE COMPOSITIONS

In one aspect, the present invention provides a compound having thestructure of Formula (I):

TM-Ln-AM  (I),

wherein TM is a targeting moiety, AM is an activating moiety that iscapable of activating a human dendritic cell, NK cell, or tumor cell, ora combination thereof, Ln is a linker, and n is an integer selected from0 and 1.

In another aspect, the present invention provides a compound having thestructure of Formula (I):

TM-Ln-AM  (I),

wherein TM is a targeting moiety, AM is an activating moiety that iscapable of binding specifically to human TLR7 and/or TLR8, Ln is alinker, and n is an integer selected from 0 and 1.

In a further aspect, the present invention provides a compound havingthe structure of Formula (I):

TM-Ln-AM  (I),

wherein TM is a targeting moiety, AM is an activating moiety, Ln is alinker, and n is an integer selected from 0 and 1, with the proviso thatwhen TM comprises an antibody, AM does not comprise a CpGoligonucleotide or a therapeutic agent.

A. Targeting Moiety

In general, compounds of the present invention comprise a target moiety.

By “targeting moiety (TM)” or “targeting agent” here in is meant amolecule, complex, or aggregate, that binds specifically or selectivelyto a target molecule, cell, particle, tissue or aggregate, whichgenerally is referred to as a “target” or a “marker,” and these arediscussed in further detail herein.

In some embodiments, the targeting moiety comprises an immunoglobulin, aprotein, a peptide, a small molecule, a nanoparticle, or a nucleic acid.

Exemplary targeting agents such as antibodies (e.g., chimeric, humanizedand human), ligands for receptors, lecitins, and saccharides, andsubstrate for certain enzymes are recognized in the art and are usefulwithout limitation in practicing the present invention. Other targetingagents include a class of compounds that do not include specificmolecular recognition motifs include nanoparticles, macromolecules suchas poly(ethylene glycol), polysaccharide, and polyamino acids which addmolecular mass to the activating moiety. The additional molecular massaffects the pharmacokinetics of the activating moiety, e.g., serumhalf-life.

In some embodiments, a targeting moiety is an antibody, antibodyfragment, bispecific antibody or other antibody-based molecule orcompound. However, other examples of targeting moieties are known in theart and may be used, such as aptamers, avimers, receptor-bindingligands, nucleic acids, biotin-avidin binding pairs, binding peptides orproteins, etc. The terms “targeting moiety” and “binding moiety” areused synonymously herein.

Targets

By “target” or “marker” herein is meant any entity that is capable ofspecifically binding to a particular targeting moiety. In someembodiments, targets are specifically associated with one or moreparticular cell or tissue types. In some embodiments, targets arespecifically associated with one or more particular disease states. Insome embodiments, targets are specifically associated with one or moreparticular developmental stages. For example, a cell type specificmarker is typically expressed at levels at least 2 fold greater in thatcell type than in a reference population of cells. In some embodiments,the cell type specific marker is present at levels at least 3 fold, atleast 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, atleast 8 fold, at least 9 fold, at least 10 fold, at least 50 fold, atleast 100 fold, or at least 1,000 fold greater than its averageexpression in a reference population. Detection or measurement of a celltype specific marker may make it possible to distinguish the cell typeor types of interest from cells of many, most, or all other types. Insome embodiments, a target can comprise a protein, a carbohydrate, alipid, and/or a nucleic acid, as described herein.

A substance is considered to be “targeted” for the purposes describedherein if it specifically binds to a nucleic acid targeting moiety. Insome embodiments, a nucleic acid targeting moiety specifically binds toa target under stringent conditions. An inventive complex or compoundcomprising targeting moiety is considered to be “targeted” if thetargeting moiety specifically binds to a target, thereby delivering theentire complex or compound composition to a specific organ, tissue,cell, extracellular matrix component, and/or intracellular compartment.

In certain embodiments, compound in accordance with the presentinvention comprise a targeting moiety which specifically binds to one ormore targets (e.g. antigens) associated with an organ, tissue, cell,extracellular matrix component, and/or intracellular compartment. Insome embodiments, compounds comprise a targeting moiety whichspecifically binds to targets associated with a particular organ ororgan system. In some embodiments, compounds in accordance with thepresent invention comprise a nuclei targeting moiety which specificallybinds to one or more intracellular targets (e.g. organelle,intracellular protein). In some embodiments, compounds comprise atargeting moiety which specifically binds to targets associated withdiseased organs, tissues, cells, extracellular matrix components, and/orintracellular compartments. In some embodiments, compounds comprise atargeting moiety which specifically binds to targets associated withparticular cell types (e.g. endothelial cells, cancer cells, malignantcells, prostate cancer cells, etc.).

In some embodiments, compounds in accordance with the present inventioncomprise a targeting moiety which binds to a target that is specific forone or more particular tissue types (e.g. liver tissue vs. prostatetissue). In some embodiments, compounds in accordance with the presentinvention comprise a targeting moiety which binds to a target that isspecific for one or more particular cell types (e.g. T cells vs. Bcells). In some embodiments, compounds in accordance with the presentinvention comprise a targeting moiety which binds to a target that isspecific for one or more particular disease states (e.g. tumor cells vs.healthy cells). In some embodiments, compounds in accordance with thepresent invention comprise a targeting moiety which binds to a targetthat is specific for one or more particular developmental stages (e.g.stem cells vs. differentiated cells).

In some embodiments, a target may be a marker that is exclusively orprimarily associated with one or a few cell types, with one or a fewdiseases, and/or with one or a few developmental stages. A cell typespecific marker is typically expressed at levels at least 2 fold greaterin that cell type than in a reference population of cells which mayconsist, for example, of a mixture containing cells from a plurality(e.g., 5-10 or more) of different tissues or organs in approximatelyequal amounts. In some embodiments, the cell type specific marker ispresent at levels at least 3 fold, at least 4 fold, at least 5 fold, atleast 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, atleast 10 fold, at least 50 fold, at least 100 fold, or at least 1000fold greater than its average expression in a reference population.Detection or measurement of a cell type specific marker may make itpossible to distinguish the cell type or types of interest from cells ofmany, most, or all other types.

In some embodiments, a target comprises a protein, a carbohydrate, alipid, and/or a nucleic acid. In some embodiments, a target comprises aprotein and/or characteristic portion thereof, such as a tumor-marker,integrin, cell surface receptor, transmembrane protein, intercellularprotein, ion channel, membrane transporter protein, enzyme, antibody,chimeric protein, glycoprotein, etc. In some embodiments, a targetcomprises a carbohydrate and/or characteristic portion thereof, such asa glycoprotein, sugar (e.g., monosaccharide, disaccharide,polysaccharide), glycocalyx (i.e., the carbohydrate-rich peripheral zoneon the outside surface of most eukaryotic cells) etc. In someembodiments, a target comprises a lipid and/or characteristic portionthereof, such as an oil, fatty acid, glyceride, hormone, steroid (e.g.,cholesterol, bile acid), vitamin (e.g. vitamin E), phospholipid,sphingolipid, lipoprotein, etc. In some embodiments, a target comprisesa nucleic acid and/or characteristic portion thereof, such as a DNAnucleic acid; RNA nucleic acid; modified DNA nucleic acid; modified RNAnucleic acid; nucleic acid that includes any combination of DNA, RNA,modified DNA, and modified RNA.

Numerous markers are known in the art. Typical markers include cellsurface proteins, e.g., receptors. Exemplary receptors include, but arenot limited to, the transferrin receptor; LDL receptor; growth factorreceptors such as epidermal growth factor receptor family members (e.g.,EGFR, Her2, Her3, Her4) or vascular endothelial growth factor receptors,cytokine receptors, cell adhesion molecules, integrins, selectins, andCD molecules. The marker can be a molecule that is present exclusivelyor in higher amounts on a malignant cell, e.g., a tumor antigen.

Tumor Antigens

In some embodiments, the targeting moiety is capable of binding to atumor cell specifically or preferably in comparison to a non-tumor cell.

The binding of target moiety to tumor cell can be measured using assaysknown in the art.

In some embodiments, the tumor cell is of a carcinoma, a sarcoma, alymphoma, a myeloma, or a central nervous system cancer.

In some embodiments, the targeting moiety is capable of binding to atumor antigen specifically or preferably in comparison to a non-tumorantigen.

By “specifically binds” or “preferably binds” herein is meant that thebinding between two binding partners (e.g., between a targeting moietyand its binding partner) is selective for the two binding partners andcan be discriminated from unwanted or non-specific interactions. Forexample, the ability of an antigen-binding moiety to bind to a specificantigenic determinant can be measured either through an enzyme-linkedimmunosorbent assay (ELISA) or other techniques familiar to one of skillin the art, e.g. surface plasmon resonance technique (analyzed on aBIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)), andtraditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). Theterms “anti-[antigen] antibody” and “an antibody that binds to[antigen]” refer to an antibody that is capable of binding therespective antigen with sufficient affinity such that the antibody isuseful as a diagnostic and/or therapeutic agent in targeting theantigen. In some embodiments, the extent of binding of an anti-[antigen]antibody to an unrelated protein is less than about 10% of the bindingof the antibody to the antigen as measured, e.g., by a radioimmunoassay(RIA). In some embodiments, an antibody that binds to [antigen] has adissociation constant (KD) of <<100 nM, <10 nM, <1 nM, <0.1 nM, <0.01nM, or <0.001 nM (e.g. 10⁻⁸ M or less, e.g. from 10⁻⁸ M to 10⁻¹³ M,e.g., from 10⁻⁹ M to 10⁻¹³ M). It is understood that the abovedefinition is also applicable to antigen-binding moieties that bind toan antigen.

In certain specific embodiments, a target is a tumor marker. In someembodiments, a tumor marker is an antigen that is present in a tumorthat is not present in normal organs, tissues, and/or cells. In someembodiments, a tumor marker is an antigen that is more prevalent in atumor than in normal organs, tissues, and/or cells. In some embodiments,a tumor marker is an antigen that is more prevalent in malignant cancercells than in normal cells.

By “tumor antigen” herein is meant an antigenic substance produced intumor cells, i.e., it triggers an immune response in the host. Normalproteins in the body are not antigenic because of self-tolerance, aprocess in which self-reacting cytotoxic T lymphocytes (CTLs) andautoantibody-producing B lymphocytes are culled “centrally” in primarylymphatic tissue (BM) and “peripherally” in secondary lymphatic tissue(mostly thymus for T-cells and spleen/lymph nodes for B cells). Thus anyprotein that is not exposed to the immune system triggers an immuneresponse. This may include normal proteins that are well sequesteredfrom the immune system, proteins that are normally produced in extremelysmall quantities, proteins that are normally produced only in certainstages of development, or proteins whose structure is modified due tomutation.

In some embodiments, a target is preferentially expressed in tumortissues and/or cells versus normal tissues and/or cells.

In some embodiments of the invention a marker is a tumor marker. Themarker may be a polypeptide that is expressed at higher levels ondividing than on non-dividing cells. For example, Her-2/neu (also knownas ErbB-2) is a member of the EGF receptor family and is expressed onthe cell surface of tumors associated with breast cancer. Anotherexample is a peptide known as F3 that is a suitable targeting agent fordirecting a nanoparticle to nucleolin (Porkka et al., 2002, Proc. Natl.Acad. Sci., USA, 99:7444; and Christian et al., 2003, J. Cell Biol.,163:871). It has been shown that targeted particles comprising ananoparticle and the A10 aptamer (which specifically binds to PSMA) wereable to specifically and effectively deliver docetaxel to prostatecancer tumors.

Antibodies or other drug that specifically target these tumor targetsspecifically interfere with and regulate signaling pathways of thebiological behavior of tumor cells regulate directly, or block signalingpathway to inhibit tumor cell growth or induce apoptosis. To date, thereare dozens of target drugs have been approved for solid tumors orhematological malignancies clinical research and treatment, and thereare number of targeted drugs for hematological malignancies.

In some embodiments, the tumor antigen (or tumor target) is selectedfrom the group consisting of: CD2, CD19, CD20, CD22, CD27, CD33, CD37,CD38, CD40, CD44, CD47, CD52, CD56, CD70, CD79, and CD137.

In some embodiments, the tumor antigen (or tumor target) is selectedfrom the group consisting of: 4-1BB, 5T4, AGS-5, AGS-16, Angiopoietin 2,B7.1, B7.2, B7DC, B7H1, B7H2, B7H3, BT-062, BTLA, CAIX, Carcinoembryonicantigen, CTLA4, Cripto, ED-B, ErbB1, ErbB2, ErbB3, ErbB4, EGFL7, EpCAM,EphA2, EphA3, EphB2, FAP, Fibronectin, Folate Receptor, Ganglioside GM3,GD2, glucocorticoid-induced tumor necrosis factor receptor (GITR),gp100, gpA33, GPNMB, ICOS, IGF1R, Integrin αν, Integrin ανβ, KIR, LAG-3,Lewis Y antigen, Mesothelin, c-MET, MN Carbonic anhydrase IX, MUC1,MUC16, Nectin-4, NKGD2, NOTCH, OX40, OX40L, PD-1, PDL1, PSCA, PSMA,RANKL, ROR1, ROR2, SLC44A4, Syndecan-1, TACI, TAG-72, Tenascin, TIM3,TRAILR1, TRAILR2, VEGFR-1, VEGFR-2, VEGFR-3, and variants thereof. Thevariants of the tumor antigen encompass various mutants or polympormismsknown in the art and/or naturally occurred.

Antibodies

In some embodiments, the targeting moiety comprises an antibody, or afunctional fragment thereof.

By immunoglobulin” or “antibody” herein is meant a full-length (i.e.,naturally occurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an immunologically active (i.e., specifically binding)portion of an immunoglobulin molecule, like an antibody fragment. Anantibody or antibody fragment may be conjugated or otherwise derivatizedwithin the scope of the claimed subject matter. Such antibodies includeIgG1, IgG2a, IgG3, IgG4 (and IgG4 subforms), as well as IgA isotypes.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity and comprise an Fc region or aregion equivalent to the Fc region of an immunoglobulin The terms“full-length antibody”, “intact antibody”, “and “whole antibody” areused herein interchangeably to refer to an antibody having a structuresubstantially similar to a native antibody structure or having heavychains that contain an Fc region as defined herein.

By “native antibodies” herein is meant naturally occurringimmunoglobulin molecules with varying structures. For example, nativeIgG antibodies are heterotetrameric glycoproteins of about 150,000daltons, composed of two identical light chains and two identical heavychains that are disulfide-bonded. From N- to C-terminus, each heavychain has a variable region (VH), also called a variable heavy domain ora heavy chain variable domain, followed by three constant domains (CHI,CH2, and CH3), also called a heavy chain constant region. Similarly,from N- to C-terminus, each light chain has a variable region (VL), alsocalled a variable light domain or a light chain variable domain,followed by a constant light (CL) domain, also called a light chainconstant region. The light chain of an antibody may be assigned to oneof two types, called kappa (κ) and lambda (λ), based on the amino acidsequence of its constant domain.

By “antibody fragment” herein is meant a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)2, diabodies, linear antibodies, single-chain antibody molecules(e.g. scFv), single-domain antibodies, and multispecific antibodiesformed from antibody fragments. For a review of certain antibodyfragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review ofscFv fragments, see e.g. Pliickthun, in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994); see also WO 93/16185; and U.S. Pat. Nos.5,571,894 and 5,587,458. For discussion of Fab and F(ab′)2 fragmentscomprising salvage receptor binding epitope residues and havingincreased in vivo half-life, see U.S. Pat. No. 5,869,046. Diabodies areantibody fragments with two antigen-binding sites that may be bivalentor bispecific. See, for example, EP 404,097; WO 1993/01161; Hudson etal., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad SciUSA 90, 6444-6448 (1993). Triabodies and tetrabodies are also describedin Hudson et al., Nat Med 9, 129-134 (2003). Single-domain antibodiesare antibody fragments comprising all or a portion of the heavy chainvariable domain or all or a portion of the light chain variable domainof an antibody. In certain embodiments, a single-domain antibody is ahuman single-domain antibody (Domantis, Inc., Waltham, Mass.; see e.g.U.S. Pat. No. 6,248,516 B 1). Antibody fragments can be made by varioustechniques, including but not limited to proteolytic digestion of anintact antibody as well as production by recombinant host cells (e.g. E.coli or phage), as described herein.

By “antigen binding domain” herein is meant the part of an antibody thatcomprises the area which specifically binds to and is complementary topart or all of an antigen. An antigen binding domain may be provided by,for example, one or more antibody variable domains (also called antibodyvariable regions). Particularly, an antigen binding domain comprises anantibody light chain variable region (VL) and an antibody heavy chainvariable region (VH).

By “variable region” or “variable domain” herein is meant the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindtet al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007).A single VH or VL domain may be sufficient to confer antigen-bindingspecificity.

By “hypervariable region” or “HVR” herein is meant each of the regionsof an antibody variable domain which are hypervariable in sequenceand/or form structurally defined loops “hypervariable loops”).Generally, native four-chain antibodies comprise six HVRs; three in theVH (HI, H2, H3), and three in the VL (LI, L2, L3). HVRs generallycomprise amino acid residues from the hypervariable loops and/or fromthe complementarity determining regions (CDRs), the latter being ofhighest sequence variability and/or involved in antigen recognition.With the exception of CDR1 in VH, CDRs generally comprise the amino acidresidues that form the hypervariable loops. Hypervariable regions (HVRs)are also referred to as “complementarity determining regions” (CDRs),and these terms are used herein interchangeably in reference to portionsof the variable region that form the antigen binding regions. Thisparticular region has been described by Kabat et al., U.S. Dept. ofHealth and Human Services, Sequences of Proteins of ImmunologicalInterest (1983) and by Chothia et al., J Mol Biol 196:901-917 (1987),where the definitions include overlapping or subsets of amino acidresidues when compared against each other. Nevertheless, application ofeither definition to refer to a CDR of an antibody or variants thereofis intended to be within the scope of the term as defined and usedherein. The exact residue numbers which encompass a particular CDR willvary depending on the sequence and size of the CDR. Those skilled in theart can routinely determine which residues comprise a particular CDRgiven the variable region amino acid sequence of the antibody.

The antibody of the present invention can be chimeric antibodies,humanized antibodies, human antibodies, or antibody fusion proteins.

By “chimeric antibody” herein is meant a recombinant protein thatcontains the variable domains of both the heavy and light antibodychains, including the complementarity determining regions (CDRs) of anantibody derived from one species, preferably a rodent antibody, morepreferably a murine antibody, while the constant domains of the antibodymolecule are derived from those of a human antibody. For veterinaryapplications, the constant domains of the chimeric antibody may bederived from that of other species, such as a subhuman primate, cat ordog.

By “humanized antibody” herein is meant a recombinant protein in whichthe CDRs from an antibody from one species; e.g., a rodent antibody, aretransferred from the heavy and light variable chains of the rodentantibody into human heavy and light variable domains. The constantdomains of the antibody molecule are derived from those of a humanantibody. In some embodiments, specific residues of the framework regionof the humanized antibody, particularly those that are touching or closeto the CDR sequences, may be modified, for example replaced with thecorresponding residues from the original rodent, subhuman primate, orother antibody.

By “human antibody” herein is meant an antibody obtained, for example,from transgenic mice that have been “engineered” to produce specifichuman antibodies in response to antigenic challenge. In this technique,elements of the human heavy and light chain locus are introduced intostrains of mice derived from embryonic stem cell lines that containtargeted disruptions of the endogenous heavy chain and light chain loci.The transgenic mice can synthesize human antibodies specific for humanantigens, and the mice can be used to produce human antibody-secretinghybridomas. Methods for obtaining human antibodies from transgenic miceare described by Green et al, Nature Genet. 7: 13 (1994), Lonberg et al,Nature 368:856 (1994), and Taylor et al, Int. Immun 6. 579 (1994). Afully human antibody also can be constructed by genetic or chromosomaltransfection methods, as well as phage display technology, all of whichare known in the art. See for example, McCafferty et al, Nature348:552-553 (1990) for the production of human antibodies and fragmentsthereof in vitro, from immunoglobulin variable domain gene repertoiresfrom unimmunized donors. In this technique, antibody variable domaingenes are cloned in-frame into either a major or minor coat protein geneof a filamentous bacteriophage, and displayed as functional antibodyfragments on the surface of the phage particle. Because the filamentousparticle contains a single-stranded DNA copy of the phage genome,selections based on the functional properties of the antibody alsoresult in selection of the gene encoding the antibody exhibiting thoseproperties. In this way, the phage mimics some of the properties of theB cell. Phage display can be performed in a variety of formats, fortheir review, see e.g. Johnson and Chiswell, Current Opinion inStructural Biology 3:5564-571 (1993). Human antibodies may also begenerated by in vitro activated B cells. See U.S. Pat. Nos. 5,567,610and 5,229,275, which are incorporated herein by reference in theirentirety.

By “antibody fusion protein” herein is meant a recombinantly-producedantigen-binding molecule in which two or more of the same or differentnatural antibody, single-chain antibody or antibody fragment segmentswith the same or different specificities are linked. A fusion proteincomprises at least one specific binding site. Valency of the fusionprotein indicates the total number of binding arms or sites the fusionprotein has to antigen(s) or epitope(s); i.e., monovalent, bivalent,trivalent or mutlivalent. The multivalency of the antibody fusionprotein means that it can take advantage of multiple interactions inbinding to an antigen, thus increasing the avidity of binding to theantigen, or to different antigens. Specificity indicates how manydifferent types of antigen or epitope an antibody fusion protein is ableto bind; i.e., monospecific, bispecific, trispecific, multispecific.Using these definitions, a natural antibody, e.g., an IgG, is bivalentbecause it has two binding arms but is monospecific because it binds toone type of antigen or epitope. A monospecific, multivalent fusionprotein has more than one binding site for the same antigen or epitope.For example, a monospecific diabody is a fusion protein with two bindingsites reactive with the same antigen. The fusion protein may comprise amultivalent or multispecific combination of different antibodycomponents or multiple copies of the same antibody component. The fusionprotein may additionally comprise a therapeutic agent.

In some embodiments, the targeting moiety is an antibody, or antibodyfragment, that is selected based on its specificity for an antigenexpressed on a target cell, or at a target site, of interest. A widevariety of tumor-specific or other disease-specific antigens have beenidentified and antibodies to those antigens have been used or proposedfor use in the treatment of such tumors or other diseases. Theantibodies that are known in the art can be used in the compounds of theinvention, in particular for the treatment of the disease with which thetarget antigen is associated. Examples of target antigens (and theirassociated diseases) to which an antibody-linker-drug conjugate of theinvention can be targeted include: CD2, CD19, CD20, CD22, CD27, CD33,CD37, CD38, CD40, CD44, CD47, CD52, CD56, CD70, CD79, CD137, 4-1BB, 5T4,AGS-5, AGS-16, Angiopoietin 2, B7.1, B7.2, B7DC, B7H1, B7H2, B7H3,BT-062, BTLA, CAIX, Carcinoembryonic antigen, CTLA4, Cripto, ED-B,ErbB1, ErbB2, ErbB3, ErbB4, EGFL7, EpCAM, EphA2, EphA3, EphB2, FAP,Fibronectin, Folate Receptor, Ganglioside GM3, GD2,glucocorticoid-induced tumor necrosis factor receptor (GITR), gp100,gpA33, GPNMB, ICOS, IGF1R, Integrin αν, Integrin ανβ, KIR, LAG-3, LewisY, Mesothelin, c-MET, MN Carbonic anhydrase IX, MUC1, MUC16, Nectin-4,NKGD2, NOTCH, OX40, OX40L, PD-1, PDL1, PSCA, PSMA, RANKL, ROR1, ROR2,SLC44A4, Syndecan-1, TACI, TAG-72, Tenascin, TIM3, TRAILR1, TRAILR2,VEGFR-1, VEGFR-2, VEGFR-3.

In some embodiments, the antibody is selected from the group consistingof: Rituxan (rituximab), Herceptin (trastuzumab), Erbitux (cetuximab),Vectibix (Panitumumab), Arzerra (Ofatumumab), Benlysta (belimumab),Yervoy (ipilimumab), Perjeta (Pertuzumab), Tremelimumab, Nivolumab,Dacetuzumab, Urelumab, MPDL3280A, Lambrolizumab, and Blinatumomab.

Rituxan (Rituximab) is a chimeric antibody used for the treatment ofB-cell non-Hodgkin's lymphoma. It acts on the surface of B cellsexpressing the CD20 antigen that is expressed on 90% of B-cellnon-Hodgkin's lymphoma. Rituxan binds CD20 to induce B cell lysisthrough CDC and ADCC, as well as sensitize human lymphocytes that aredrug resistance for some cytotoxic chemotherapeutics.

Herceptin (Trastuzumab) is a humanized monoclonal antibody that acts onhuman epidermal growth factor receptor extracellular domain of Her2,which is expressed in 25% -30% of breast cancer. It is believed thatTrastuzumab has anti-tumor effect through (1) down-regulation Her2receptor, inhibition of Her2 intracellular signaling transductionpathways and induction of apoptosis; (2) immune mechanisms relatedantibody dependent ADCC and CDC to kill tumor cells; (3) enhance theeffects of chemotherapy.

Erbitux (Cetuximab) is a chimeric antibody that acts on epidermal growthfactor receptor (EGFR). Erbitux binds EGFR to inhibit its signaltransduction pathway, affecting cell proliferation, invasion andmetastasis, and angiogenesis. Inhibition of EGFR signal transductionpathway can enhance chemotherapy drugs and radiation therapy efficacy.

Avastin (Bevacizumab) is a humanized monoclonal antibody that targetsvascular endothelial growth factor (VEGF). Its binding of VEGFR inhibitsVEGF and signal transduction, resulting in inhibition of tumorangiogenesis.

Other antibodies that currently under development can also be used astargeting moiety. For example, therapeutic monoclonal antibodies againstthe following targets are under development for treatment of tumors:CD2, CD19, CD20, CD22, CD27, CD33, CD37, CD38, CD40, CD44, CD47, CD52,CD56, CD70, CD79, and CD137 and the following targets for treatment oftumors: 4-1BB, 5T4, AGS-5, AGS-16, Angiopoietin 2, B7.1, B7.2, B7DC,B7H1, B7H2, B7H3, BT-062, BTLA, CAIX, Carcinoembryonic antigen, CTLA4,Cripto, ED-B, ErbB1, ErbB2, ErbB3, ErbB4, EGFL7, EpCAM, EphA2, EphA3,EphB2, FAP, Fibronectin, Folate Receptor, Ganglioside GM3, GD2,glucocorticoid-induced tumor necrosis factor receptor (GITR), gp100,gpA33, GPNMB, ICOS, IGF1R, Integrin αν, Integrin ανβ, KIR, LAG-3, Lewis,Mesothelin, c-MET, MN Carbonic anhydrase IX, MUC1, MUC16, Nectin-4,NKGD2, NOTCH, OX40, OX40L, PD-1, PDL1, PSCA, PSMA, RANKL, ROR1, ROR2,SLC44A4, Syndecan-1, TACI, TAG-72, Tenascin, TIM3, TRAILR1, TRAILR2,VEGFR-1, VEGFR-2, and VEGFR-3 and their variant. (Scott A M, Wolchok JD, Old L J. Antibody Therapy of Cancer. Nat Rev Cancer. 2012 Mar. 22;12(4):278-87).

In some embodiments, the targeting moiety comprises a Fab, Fab′,F(ab′)2, single domain antibody, T and Abs dimer, Fv, scFv, dsFv,ds-scFv, Fd, linear antibody, minibody, diabody, bispecific antibodyfragment, bibody, tribody, sc-diabody, kappa (lamda) body, BiTE, DVD-Ig,SIP, SMIP, DART, or an antibody analogue comprising one or more CDRs.

The following table shows the various antibody structures and thetargets being studied.

TABLE 1 Antibody Structure Exemplary Target scFv CC49, ERBB2, LeyDiabody Ley and TAG-72 Affibody ERBB2 Minibody CEA, ERBB2 Protein-FcAngiopoietin 1, angiopoietin 2, VEGFR1, VEGER2 Intact IgG CD20, CD33,EGFR, ERBB2, VEGF IgE and IgM GM2 Drug conjugates CD30, CD33 and ERBB2Loaded nanoparticles A33, EGFR and transferrin. Bispecifics CD19-CD3,EPCAM---CD3, gp100-CD3

In some embodiments, the targeting moiety comprises a ATWLPPRpolypeptide of VEGFR, Thrombospondin-1 mimetics, CDCRGDCFCG (cyclic)polypeptide, SCH 221153 fragment, NCNGRC (cyclic) polypeptide,CTTHWGFTLC polypeptide, CGNKRTRGC polypeptide (LyP-1), Octreotide,Vapreotide, Lanreotide, C-3940 polypeptide, Decapeptyl, Lupron, Zoladex,or Cetrorelix.

Folic Acid

In some embodiments, the targeting moiety comprises folic acid or aderivative thereof.

In recent years, research on folic acid had made great progress. Folicacid is a small molecule vitamin that is necessary for cell division.Tumor cells divide abnormally and there is a high expression of folatereceptor (FR) on tumor cell surface to capture enough folic acid tosupport cell division.

Data indicate FR expression in tumor cells is 20-200 times higher thannormal cells. The expression rate of FR in various malignant tumors are:82% in ovarian cancer, 66% in non-small cell lung cancer, 64% in kidneycancer, 34% in colon cancer, and 29% in breast cancer (Xia W, Low P S.Late-targeted therapies for cancer. J Med Chem. 2010; 14; 53(19):6811-24). The expression rate of FA and the degree of malignancy ofepithelial tumor invasion and metastasis is positively correlated. FAenters cell through FR mediated endocytosis, and FA through its carboxylgroup forms FA complexes with drugs which enter the cells. Under acidicconditions (pH value of 5), FR separates from the FA, and FA releasesdrugs into the cytoplasm.

Clinically, the system can be used to deliver drugs selectively attackthe tumor cells. Folic acid has small molecular weight, hasnon-immunogenicity and high stability, and is inexpensive to synthesis.More importantly, chemical coupling between the drug and the carrier issimple, and as such using FA as targeting molecule to construct drugdelivery system has become a research hotspot for cancer treatment.Currently EC145 (FA chemotherapy drug conjugate compound) that is inclinical trials can effectively attack cancer cells (Pribble P andEdelman M J. EC145: a novel targeted agent for adenocarcinoma of thelung. Expert Opin. Investig. Drugs (2012) 21:755-761).

Other Targeting Moieties

In some embodiments, the targeting moiety comprises extracellulardomains (ECD) or soluble form of PD-1, CTLA4, CD47, BTLA, KIR, TIM3,4-1BB, and LAG3, full length of partial of a surface ligandAmphiregulin, Betacellulin, EGF, Ephrin, Epigen, Epiregulin, IGF,Neuregulin, TGF, TRAIL, or VEGF.

Nanoparticles

In some embodiments, the targeting moiety comprises a particle (targetparticle), preferably a nanoparticle, optionally a targeted nanoparticlethat attached to a targeting molecule that can binds specifically orpreferably to a target. In some embodiments, the targeting particle byitself guides the compound of the present invention (such as byenrichment in tumor cells or tissue) and there is no additionaltargeting molecules attached therein.

By “nanoparticle” herein is meant any particle having a diameter of lessthan 1000 nm. In some embodiments, a therapeutic agent and/or targetingmolecule can be associated with the polymeric matrix. In someembodiments, the targeting molecule can be covalently associated withthe surface of a polymeric matrix. In some embodiments, covalentassociation is mediated by a linker. In some embodiments, thetherapeutic agent can be associated with the surface of, encapsulatedwithin, surrounded by, and/or dispersed throughout the polymeric matrix.U.S. Pat. No. 8,246,968, which is incorporated in its entirety.

In general, nanoparticles of the present invention comprise any type ofparticle. Any particle can be used in accordance with the presentinvention. In some embodiments, particles are biodegradable andbiocompatible. In general, a biocompatible substance is not toxic tocells. In some embodiments, a substance is considered to bebiocompatible if its addition to cells results in less than a certainthreshold of cell death. In some embodiments, a substance is consideredto be biocompatible if its addition to cells does not induce adverseeffects. In general, a biodegradable substance is one that undergoesbreakdown under physiological conditions over the course of atherapeutically relevant time period (e.g., weeks, months, or years). Insome embodiments, a biodegradable substance is a substance that can bebroken down by cellular machinery. In some embodiments, a biodegradablesubstance is a substance that can be broken down by chemical processes.In some embodiments, a particle is a substance that is bothbiocompatible and biodegradable. In some embodiments, a particle is asubstance that is biocompatible, but not biodegradable. In someembodiments, a particle is a substance that is biodegradable, but notbiocompatible.

In some embodiments, particles are greater in size than the renalexcretion limit (e.g. particles having diameters of greater than 6 nm).In some embodiments, particles are small enough to avoid clearance ofparticles from the bloodstream by the liver (e.g. particles havingdiameters of less than 1000 nm). In general, physiochemical features ofparticles should allow a targeted particle to circulate longer in plasmaby decreasing renal excretion and liver clearance.

It is often desirable to use a population of particles that isrelatively uniform in terms of size, shape, and/or composition so thateach particle has similar properties. For example, at least 80%, atleast 90%, or at least 95% of the particles may have a diameter orgreatest dimension that falls within 5%, 10%, or 20% of the averagediameter or greatest dimension. In some embodiments, a population ofparticles may be heterogeneous with respect to size, shape, and/orcomposition.

Zeta potential is a measurement of surface potential of a particle. Insome embodiments, particles have a zeta potential ranging between −50 mVand +50 mV. In some embodiments, particles have a zeta potential rangingbetween −25 mV and +25 mV. In some embodiments, particles have a zetapotential ranging between −10 mV and +10 mV. In some embodiments,particles have a zeta potential ranging between −5 mV and +5 mV. In someembodiments, particles have a zeta potential ranging between 0 mV and+50 mV. In some embodiments, particles have a zeta potential rangingbetween 0 mV and +25 mV. In some embodiments, particles have a zetapotential ranging between 0 mV and +10 mV. In some embodiments,particles have a zeta potential ranging between 0 mV and +5 mV. In someembodiments, particles have a zeta potential ranging between −50 mV and0 mV. In some embodiments, particles have a zeta potential rangingbetween −25 mV and 0 mV. In some embodiments, particles have a zetapotential ranging between −10 mV and 0 mV. In some embodiments,particles have a zeta potential ranging between −5 mV and 0 mV. In someembodiments, particles have a substantially neutral zeta potential (i.e.approximately 0 mV).

A variety of different particles can be used in accordance with thepresent invention. In some embodiments, particles are spheres orspheroids. In some embodiments, particles are spheres or spheroids. Insome embodiments, particles are flat or plate-shaped. In someembodiments, particles are cubes or cuboids. In some embodiments,particles are ovals or ellipses. In some embodiments, particles arecylinders, cones, or pyramids.

In some embodiments, particles are microparticles (e.g. microspheres).In general, a “microparticle” refers to any particle having a diameterof less than 1000 μm. In some embodiments, particles are picoparticles(e.g. picospheres). In general, a “picoparticle” refers to any particlehaving a diameter of less than 1 nm. In some embodiments, particles areliposomes. In some embodiments, particles are micelles.

Particles can be solid or hollow and can comprise one or more layers(e.g., nanoshells, nanorings). In some embodiments, each layer has aunique composition and unique properties relative to the other layer(s).For example, particles may have a core/shell structure, wherein the coreis one layer and the shell is a second layer. Particles may comprise aplurality of different layers. In some embodiments, one layer may besubstantially cross-linked, a second layer is not substantiallycross-linked, and so forth. In some embodiments, one, a few, or all ofthe different layers may comprise one or more therapeutic or diagnosticagents to be delivered. In some embodiments, one layer comprises anagent to be delivered, a second layer does not comprise an agent to bedelivered, and so forth. In some embodiments, each individual layercomprises a different agent or set of agents to be delivered.

In some embodiments, a particle is porous, by which is meant that theparticle contains holes or channels, which are typically small comparedwith the size of a particle. For example a particle may be a poroussilica particle, e.g., a mesoporous silica nanoparticle or may have acoating of mesoporous silica (Lin et al., 2005, J. Am. Chem. Soc.,17:4570). Particles may have pores ranging from about 1 nm to about 50nm in diameter, e.g., between about 1 and 20 nm in diameter. Betweenabout 10% and 95% of the volume of a particle may consist of voidswithin the pores or channels.

Particles may have a coating layer. Use of a biocompatible coating layercan be advantageous, e.g., if the particles contain materials that aretoxic to cells. Suitable coating materials include, but are not limitedto, natural proteins such as bovine serum albumin (BSA), biocompatiblehydrophilic polymers such as polyethylene glycol (PEG) or a PEGderivative, phospholipid-(PEG), silica, lipids, polymers, carbohydratessuch as dextran, other nanoparticles that can be associated withinventive nanoparticles etc. Coatings may be applied or assembled in avariety of ways such as by dipping, using a layer-by-layer technique, byself-assembly, conjugation, etc. Self-assembly refers to a process ofspontaneous assembly of a higher order structure that relies on thenatural attraction of the components of the higher order structure(e.g., molecules) for each other. It typically occurs through randommovements of the molecules and formation of bonds based on size, shape,composition, or chemical properties.

Examples of polymers include polyalkylenes (e.g. polyethylenes),polycarbonates (e.g. poly(1,3-dioxan-2one)), polyanhydrides (e.g.poly(sebacic anhydride)), polyhydroxyacids (e.g.poly((3-hydroxyalkanoate)), polyfumarates, polycaprolactones, polyamides(e.g. polycaprolactam), polyacetals, polyethers, polyesters (e.g.polylactide, polyglycolide), poly(orthoesters), polyvinyl alcohols,polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates,polycyanoacrylates, polyureas, polystyrenes, and polyamines. In someembodiments, polymers in accordance with the present invention includepolymers which have been approved for use in humans by the U.S. Food andDrug Administration (FDA) under 21 C.F.R. §177.2600, including but notlimited to polyesters (e.g. polylactic acid, polyglycolic acid,poly(lactic-co-glycolic acid), polycaprolactone, polyvalerolactone,poly(1,3-dioxan-2one)); polyanhydrides (e.g. poly(sebacic anhydride));polyethers (e.g., polyethylene glycol); polyurethanes;polymethacrylates; polyacrylates; and polycyanoacrylates.

In some embodiments, particles can be non-polymeric particles (e.g.metal particles, quantum dots, ceramic particles, polymers comprisinginorganic materials, bone-derived materials, bone substitutes, viralparticles, etc.). In some embodiments, a therapeutic or diagnostic agentto be delivered can be associated with the surface of such anon-polymeric particle. In some embodiments, a non-polymeric particle isan aggregate of non-polymeric components, such as an aggregate of metalatoms (e.g. gold atoms). In some embodiments, a therapeutic ordiagnostic agent to be delivered can be associated with the surface ofand/or encapsulated within, surrounded by, and/or dispersed throughoutan aggregate of non-polymeric components.

Particles (e.g. nanoparticles, microparticles) may be prepared using anymethod known in the art. For example, particulate formulations can beformed by methods as nanoprecipitation, flow focusing fluidic channels,spray drying, single and double emulsion solvent evaporation, solventextraction, phase separation, milling, microemulsion procedures,microfabrication, nanofabrication, sacrificial layers, simple andcomplex coacervation, and other methods well known to those of ordinaryskill in the art. Alternatively or additionally, aqueous and organicsolvent syntheses for monodisperse semiconductor, conductive, magnetic,organic, and other nanoparticles have been described (Pellegrino et al.,2005, Small, 1:48; Murray et al., 2000, Ann. Rev. Mat. Sci., 30:545; andTrindade et al., 2001, Chem. Mat., 13:3843).

Methods for making microparticles for delivery of encapsulated agentsare described in the literature (see, e.g., Doubrow, Ed., “Microcapsulesand Nanoparticles in Medicine and Pharmacy,” CRC Press, Boca Raton,1992; Mathiowitz et al., 1987, J. Control. Release, 5:13; Mathiowitz etal., 1987, Reactive Polymers, 6: 275; and Mathiowitz et al., 1988, J.Appl. Polymer Sci., 35:755).

Nucleic Acid Targeting Moieties

In some embodiments, the targeting moiety comprises an nucleic acidtargeting moiety.

In general, a nucleic acid targeting moiety is any polynucleotide thatbinds to a component associated with an organ, tissue, cell,extracellular matrix component, and/or intracellular compartment (thetarget).

In some embodiments, nucleic acid targeting moieties are aptamers.

An aptamer is typically a polynucleotide that binds to a specific targetstructure that is associated with a particular organ, tissue, cell,extracellular matrix component, and/or intracellular compartment. Ingeneral, the targeting function of the aptamer is based on thethree-dimensional structure of the aptamer. In some embodiments, bindingof an aptamer to a target is typically mediated by the interactionbetween the two- and/or three-dimensional structures of both the aptamerand the target. In some embodiments, binding of an aptamer to a targetis not solely based on the primary sequence of the aptamer, but dependson the three-dimensional structure(s) of the aptamer and/or target. Insome embodiments, aptamers bind to their targets via complementaryWatson-Crick base pairing which is interrupted by structures (e.g.hairpin loops) that disrupt base pairing.

In some embodiments, nucleic acid targeting moieties are spiegelmers(PCT Publications WO 98/08856, WO 02/100442, and WO 06/117217). Ingeneral, spiegelmers are synthetic, mirror-image nucleic acids that canspecifically bind to a target (i.e. mirror image aptamers). Spiegelmersare characterized by structural features which make them not susceptibleto exo- and endo-nucleases.

One of ordinary skill in the art will recognize that any nucleic acidtargeting moiety (e.g. aptamer or spiegelmer) that is capable ofspecifically binding to a target can be used in accordance with thepresent invention. In some embodiments, nucleic acid targeting moietiesto be used in accordance with the present invention may target a markerassociated with a disease, disorder, and/or condition. In someembodiments, nucleic acid targeting moieties to be used in accordancewith the present invention may target cancer-associated targets. In someembodiments, nucleic acid targeting moieties to be used in accordancewith the present invention may target tumor markers. Any type of cancerand/or any tumor marker may be targeted using nucleic acid targetingmoieties in accordance with the present invention. To give but a fewexamples, nucleic acid targeting moieties may target markers associatedwith prostate cancer, lung cancer, breast cancer, colorectal cancer,bladder cancer, pancreatic cancer, endometrial cancer, ovarian cancer,bone cancer, esophageal cancer, liver cancer, stomach cancer, braintumors, cutaneous melanoma, and/or leukemia.

Nucleic acids of the present invention (including nucleic acid nucleicacid targeting moieties and/or functional RNAs to be delivered, e.g.,RNAi-inducing entities, ribozymes, tRNAs, etc., described in furtherdetail below) may be prepared according to any available techniqueincluding, but not limited to chemical synthesis, enzymatic synthesis,enzymatic or chemical cleavage of a longer precursor, etc. Methods ofsynthesizing RNAs are known in the art (see, e.g., Gait, M. J. (ed.)Oligonucleotide synthesis: a practical approach, Oxford [Oxfordshire],Washington, D.C.: IRL Press, 1984; and Herdewijn, P. (ed.)Oligonucleotide synthesis: methods and applications, Methods inmolecular biology, v. 288 (Clifton, N.J.) Totowa, N.J.: Humana Press,2005).

The nucleic acid that forms the nucleic acid nucleic acid targetingmoiety may comprise naturally occurring nucleosides, modifiednucleosides, naturally occurring nucleosides with hydrocarbon linkers(e.g., an alkylene) or a polyether linker (e.g., a PEG linker) insertedbetween one or more nucleosides, modified nucleosides with hydrocarbonor PEG linkers inserted between one or more nucleosides, or acombination of thereof. In some embodiments, nucleotides or modifiednucleotides of the nucleic acid nucleic acid targeting moiety can bereplaced with a hydrocarbon linker or a polyether linker provided thatthe binding affinity and selectivity of the nucleic acid nucleic acidtargeting moiety is not substantially reduced by the substitution (e.g.,the dissociation constant of the nucleic acid nucleic acid targetingmoiety for the target should not be greater than about 1×10⁻³ M).

It will be appreciated by those of ordinary skill in the art thatnucleic acids in accordance with the present invention may comprisenucleotides entirely of the types found in naturally occurring nucleicacids, or may instead include one or more nucleotide analogs or have astructure that otherwise differs from that of a naturally occurringnucleic acid. U.S. Pat. Nos. 6,403,779; 6,399,754; 6,225,460; 6,127,533;6,031,086; 6,005,087; 5,977,089; and references therein disclose a widevariety of specific nucleotide analogs and modifications that may beused. See Crooke, S. (ed.) Antisense Drug Technology: Principles,Strategies, and Applications (1st ed), Marcel Dekker; ISBN: 0824705661;1st edition (2001) and references therein. For example, 2′-modificationsinclude halo, alkoxy and allyloxy groups. In some embodiments, the 2′-OHgroup is replaced by a group selected from H, OR, R, halo, SH, SR, NH2,NHR, NR2 or CN, wherein R is C1-C6 alkyl, alkenyl, or alkynyl, and halois F, Cl, Br, or I. Examples of modified linkages includephosphorothioate and 5′-N-phosphoramidite linkages.

Nucleic acids comprising a variety of different nucleotide analogs,modified backbones, or non-naturally occurring internucleoside linkagescan be utilized in accordance with the present invention. Nucleic acidsof the present invention may include natural nucleosides (i.e.,adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine,deoxythymidine, deoxyguanosine, and deoxycytidine) or modifiednucleosides. Examples of modified nucleotides include base modifiednucleoside (e.g., aracytidine, inosine, isoguanosine, nebularine,pseudouridine, 2,6-diaminopurine, 2-aminopurine, 2-thiothymidine,3-deaza-5-azacytidine, 2′-deoxyuridine, 3-nitropyrrole, 4-methylindole,4-thiouridine, 4-thiothymidine, 2-aminoadenosine, 2-thiothymidine,2-thiouridine, 5-bromocytidine, 5-iodouridine, inosine, 6-azauridine,6-chloropurine, 7-deazaadenosine, 7-deazaguanosine, 8-azaadenosine,8-azidoadenosine, benzimidazole, M1-methyladenosine, pyrrolo-pyrimidine,2-amino-6-chloropurine, 3-methyl adenosine, 5-propynylcytidine,5-propynyluridine, 5-bromouridine, 5-fluorouridine, 5-methylcytidine,7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine,O(6)-methylguanine, and 2-thiocytidine), chemically or biologicallymodified bases (e.g., methylated bases), modified sugars (e.g.,2′-fluororibose, 2′-aminoribose, 2′-azidoribose, 2′-O-methylribose,L-enantiomeric nucleosides arabinose, and hexose), modified phosphategroups (e.g., phosphorothioates and 5′-N-phosphoramidite linkages), andcombinations thereof. Natural and modified nucleotide monomers for thechemical synthesis of nucleic acids are readily available. In somecases, nucleic acids comprising such modifications display improvedproperties relative to nucleic acids consisting only of naturallyoccurring nucleotides. In some embodiments, nucleic acid modificationsdescribed herein are utilized to reduce and/or prevent digestion bynucleases (e.g. exonucleases, endonucleases, etc.). For example, thestructure of a nucleic acid may be stabilized by including nucleotideanalogs at the 3′ end of one or both strands order to reduce digestion.

Modified nucleic acids need not be uniformly modified along the entirelength of the molecule. Different nucleotide modifications and/orbackbone structures may exist at various positions in the nucleic acid.One of ordinary skill in the art will appreciate that the nucleotideanalogs or other modification(s) may be located at any position(s) of anucleic acid such that the function of the nucleic acid is notsubstantially affected. To give but one example, modifications may belocated at any position of a nucleic acid targeting moiety such that theability of the nucleic acid targeting moiety to specifically bind to thetarget is not substantially affected. The modified region may be at the5′-end and/or the 3′-end of one or both strands. For example, modifiednucleic acid targeting moieties in which approximately 1-5 residues atthe 5′ and/or 3′ end of either of both strands are nucleotide analogsand/or have a backbone modification have been employed. The modificationmay be a 5′ or 3′ terminal modification. One or both nucleic acidstrands may comprise at least 50% unmodified nucleotides, at least 80%unmodified nucleotides, at least 90% unmodified nucleotides, or 100%unmodified nucleotides.

Nucleic acids in accordance with the present invention may, for example,comprise a modification to a sugar, nucleoside, or internucleosidelinkage such as those described in U.S. Patent Application Publications2003/0175950, 2004/0192626, 2004/0092470, 2005/0020525, and2005/0032733. The present invention encompasses the use of any nucleicacid having any one or more of the modification described therein. Forexample, a number of terminal conjugates, e.g., lipids such ascholesterol, lithocholic acid, aluric acid, or long alkyl branchedchains have been reported to improve cellular uptake. Analogs andmodifications may be tested using, e.g., using any appropriate assayknown in the art, for example, to select those that result in improveddelivery of a therapeutic or diagnostic agent, improved specific bindingof an nucleic acid targeting moiety to a target, etc. In someembodiments, nucleic acids in accordance with the present invention maycomprise one or more non-natural nucleoside linkages. In someembodiments, one or more internal nucleotides at the 3′-end, 5′-end, orboth 3′- and 5′-ends of the nucleic acid targeting moiety are invertedto yield a linkage such as a 3′-3′ linkage or a 5′-5′ linkage.

In some embodiments, nucleic acids in accordance with the presentinvention are not synthetic, but are naturally-occurring entities thathave been isolated from their natural environments.

Any method can be used to design novel nucleic acid targeting moieties(see, e.g., U.S. Pat. Nos. 6,716,583; 6,465,189; 6,482,594; 6,458,543;6,458,539; 6,376,190; 6,344,318; 6,242,246; 6,184,364; 6,001,577;5,958,691; 5,874,218; 5,853,984; 5,843,732; 5,843,653; 5,817,785;5,789,163; 5,763,177; 5,696,249; 5,660,985; 5,595,877; 5,567,588; and5,270,163; and U.S. Patent Application Publications 2005/0069910,2004/0072234, 2004/0043923, 2003/0087301, 2003/0054360, and2002/0064780). The present invention provides methods for designingnovel nucleic acid targeting moieties. The present invention furtherprovides methods for isolating or identifying novel nucleic acidtargeting moieties from a mixture of candidate nucleic acid targetingmoieties.

Nucleic acid targeting moieties that bind to a protein, a carbohydrate,a lipid, and/or a nucleic acid can be designed and/or identified. Insome embodiments, nucleic acid targeting moieties can be designed and/oridentified for use in the complexes of the invention that bind toproteins and/or characteristic portions thereof, such as tumor-markers,integrins, cell surface receptors, transmembrane proteins, intercellularproteins, ion channels, membrane transporter proteins, enzymes,antibodies, chimeric proteins etc. In some embodiments, nucleic acidtargeting moieties can be designed and/or identified for use in thecomplexes of the invention that bind to carbohydrates and/orcharacteristic portions thereof, such as glycoproteins, sugars (e.g.,monosaccharides, disaccharides and polysaccharides), glycocalyx (i.e.,the carbohydrate-rich peripheral zone on the outside surface of mosteukaryotic cells) etc. In some embodiments, nucleic acid targetingmoieties can be designed and/or identified for use in the complexes ofthe invention that bind to lipids and/or characteristic portionsthereof, such as oils, saturated fatty acids, unsaturated fatty acids,glycerides, hormones, steroids (e.g., cholesterol, bile acids), vitamins(e.g. vitamin E), phospholipids, sphingolipids, lipoproteins etc. Insome embodiments, nucleic acid targeting moieties can be designed and/oridentified for use in the complexes of the invention that bind tonucleic acids and/or characteristic portions thereof, such as DNAnucleic acids; RNA nucleic acids; modified DNA nucleic acids; modifiedRNA nucleic acids; and nucleic acids that include any combination ofDNA, RNA, modified DNA, and modified RNA; etc.

Nucleic acid targeting moieties (e.g. aptamers or spiegelmers) may bedesigned and/or identified using any available method. In someembodiments, nucleic acid targeting moieties are designed and/oridentified by identifying nucleic acid targeting moieties from acandidate mixture of nucleic acids. Systemic Evolution of Ligands byExponential Enrichment (SELEX), or a variation thereof, is a commonlyused method of identifying nucleic acid targeting moieties that bind toa target from a candidate mixture of nucleic acids.

Nucleic acid targeting moieties that bind selectively to any target canbe isolated by the SELEX process, or a variation thereof, provided thatthe target can be used as a target in the SELEX process.

B. Activating Moiety

In general, the compound of the present invention comprises anactivating moiety.

By “activating moiety” herein is meant a molecule or agent that iscapable of stimulating or enhancing the body's immune system or tumorcells. In general, the activating moiety acts, directly or indirectly,on toll like receptors, nucleotide-oligomerization domain-likereceptors, RIG-I-Like receptors, c-type lectin receptors, or cytosolicDNA Sensors, or a combination thereof.

In some embodiments, the activating moiety is capable of activatinghuman immune cells, including but not limited to dendritic cells,macrophages, monocytes, myeloid-derived suppressor cells, NK cells, Bcells, T cells, or tumor cells, or a combination thereof.

Dendritic Cells

Dendritic cells are the most powerful antigen-presenting cells.Dendritic cells play an essential role for the initiation of both innateand adaptive immune responses. Dendritic cells also play a key role inthe induction and maintenance of immune tolerance.

By “dendritic cells” (DC) herein is meant a heterogeneous cellpopulation including two main subtypes: namely, myeloid DC (mDC) andplasmacytoid DC (pDC) (Steinman et al., 1979, J. Exp. Med., 149, 1-16).These two blood DC subsets were originally differentiated by theirexpression of CD11c (integrin complement receptor) and CD123 (IL-3Rα).Each of the pDC and mDC populations constitutes between about 0.2 toabout 0.6% of the PBMC population in humans.

By “pDC” herein is meant plasmacytoid dendritic cells and they representa subtype of dendritic cells found in the blood and peripheral lymphoidorgans. These cells express the surface markers CD123, BDCA-2(CD303) andBDCA-4(CD304) and HLA-DR, but do not express CD11c, CD14, CD3, CD20 orCD56, which distinguishes them from conventional dendritic cells,monocytes, T-cells, B cells and NK cells. As components of the innateimmune system, these cells express intracellular Toll-like receptors 7and 9, which enable the detection of viral and bacterial nucleic acids,such as ssRNA or CpG DNA motifs. Upon stimulation and subsequentactivation, these cells produce large amounts of Type I interferon(mainly IFN-α and IFN-β) and Type III interferon (e.g., IFN-X), whichare critical pleiotropic anti-viral compounds mediating a wide range ofeffects. By generating a large number of type I interferon, cytokinesand chemokines, plasmacytoid dendritic cells are widely involved in thebody's innate and adaptive immune responses. They can regulate NK cells,T cells, B cells and other cells involved in immune response intensity,duration, and response mode, thus play a very important function intumor, infection and autoimmune disease. (Liu Y J. IPC: professionaltype 1 interferon-producing cells and plasmacytoid dendritic cellprecursors. Annu Rev Immunol. 2005; 23:275-306. Gilliet M, Cao W, Liu YJ. Plasmacytoid dendritic cells: sensing nucleic acids in viralinfection and autoimmune diseases. Nat Rev Immunol. 2008 August; 8(8):594-606).

By “mDC” herein is meant myeloid dendritic cells and they represent asubtype of circulating dendritic cells found in blood and peripherallymphoid organs. These cells express the surface markers CD11c, CD1a,HLA-DR and either BDCA-1 (CD1c) or BDCA-3 (CD141). They do not expressBDCA-2 or CD123, which distinguishes them from pDC. mDC also do notexpress CD3, CD20 or CD56. As components of the innate immune system,mDC express Toll-like receptors (TLR), including TLR2, 3, 4, 5, 6 and 8,which enable the detection of bacterial and viral components. Uponstimulation and subsequent activation, these cells are the most potentantigen presenting cells to activate antigen-specific CD4 as well as CD8T cells. In addition, mDCs has the ability to produce large amounts ofIL-12 and IL23, which is critical for the induction of Th1-mediated orTh17 cell-mediated immunity

Study found that many solid tumors such as breast cancer and head andneck cancer, ovarian cancer has pDC's invasion (Treilleux I, Blay J Y,Bendriss-Vermare N et al. Dendritic cell infiltration and prognosis ofearly stage breast cancer. Clin Cancer Res 2004; 10:7466-7474. HartmannE, Wollenberg B, Rothenfusser S et al. Identification and functionalanalysis of tumor-infiltrating plasmacytoid dendritic cells in head andneck cancer. Cancer Res 2003; 63:6478-6487. Zou W P, Machelon V,Coulomb-L'Hermin A, et al. Stromal-derived factor-1 in human tumorsrecruits and alters the function of plasmacytoid precursor dendriticcells. Nat Med 2001; 7:1339-1346) and factors secreted by tumor cellsinhibit DC maturation. (Gabrilovich D I, Corak J, Ciernik I F et al.Decreased antigen presentation by dendritic cells in patients withbreast cancer. Clin Cancer Res 1997; 3:483-490. Bell D, Chomarat P,Broyles D et al. In breast carcinoma tissue, immature dendritic cellsreside within the tumor, whereas mature dendritic cells are located inperitumoral areas. J Exp Med 1999; 190:1417-1425. Menetrier-Caux C,Montmain G, Dieu M C et al Inhibition of the differentiation ofdendritic cells from CD34 (+) progenitors by tumor cells: role ofinterleukin-6 and macrophage colony-stimulating factor. Blood 1998;92:4778-4791). These immature DC cells did not play a role in promotinganti-tumor immunity. By contrast, DCs within the tumor microenvironmentpromote tumor growth by inhibiting antitumor immunity and by promotingangiogenesis. There is evidence that Toll-like receptor 7 agonistImiquimod, and Toll-like receptor 9 agonist CpG drugs can stimulate pDCwithin the tumor microenvironment to inhibit tumor development. (DummerR, Urosevic M, Kempf W et al. Imiquimod in basal cell carcinoma: howdoes it work? Br J Dermatol 2003; 149:57-58. Miller R L, Gerster J F,Owens M L et al Imiquimod applied topically: a novel immune responsemodifier and new class of drug. Int J Immunopharmacol 1999; 21:1-14.Hofmann M A, Kors C, Audring H et al Phase 1 evaluation ofintralesionally injected TLR9-agonist PF-3512676 in patients with basalcell carcinoma or metastatic melanoma. J Immunother 2008; 31:520-527).

In some embodiments, the human dendritic cell is a plasmacytoiddendritic cell.

In some embodiments, the human dendritic cell is a myeloid dendriticcell.

In some embodiments, the activating moiety is capable of bindingspecifically to human TLR7 or TLR8 and thus is capable of activatingeither pDC or mDC.

In some embodiments, the activating moiety is capable of bindingspecifically to both human TLR7 and TLR8, thus is capable of activatingboth pDC and mDC. pDCs selectively express endosomal Toll-like receptor(TLR) 7 and TLR9, whereas mDCs selectively express TLR8. Bao M, Liu Y J.Regulation of TLR7/9 signaling in plasmacytoid dendritic cells, ProteinCel. 2013 January; 4(1):40-52. Hémont C, Neel A, Heslan M, Braudeau C,Josien R. Human blood mDC subsets exhibit distinct TLR repertoire andresponsiveness. J Leukoc Biol. 2013 April; 93(4):599-609.

Natural Killer Cells (NK Cells)

In some embodiments, the activating moiety is capable of activatingnatural killer cells (NK cells), preferably human NK cells.

Natural killer (NK) cells are a type of cytotoxic lymphocyte thatconstitutes a major component of the immune system. NK cells are asubset of peripheral blood lymphocytes defined by the expression of CD56or CD16 and the absence of the T cell receptor (CD3). They recognize andkill transformed cell lines without priming in an MHC-unrestrictedfashion. NK cells play a major role in the rejection of tumors and cellsinfected by viruses. The process by which an NK cell recognizes a targetcell and delivers a sufficient signal to trigger target lysis isdetermined by an array of inhibitory and activating receptors on thecell surface. NK discrimination of self from altered self involvesinhibitory receptor recognition of MHC-I molecules and non-MHC ligandslike CD48 and Clr-1b. NK recognition of infected or damaged cells(altered self) is coordinated through stress induced ligands (e.g.,MICA, MICB, Rae1, H60, Mult1) or virally encoded ligands (e.g., m157,hemagluttinin) recognized by various activating receptors, includingNKG2D, Ly49H and NKp46/Ncr1.

NK cells represent the predominant lymphoid cell in the peripheral bloodfor many months after allogeneic or autologous stem cell transplant andthey have a primary role in immunity to pathogens during this period(Reittie et al (1989) Blood 73: 1351-1358; Lowdell et al (1998) BoneMarrow Transplant 21: 679-686). The role of NK cells in engraftment,graft-versus-host disease, anti-leukemia activity and post-transplantinfection is reviewed in Lowdell (2003) Transfusion Medicine 13:399-404.

Human NK cells mediate the lysis of tumor cells and virus-infected cellsvia natural cytotoxicity and antibody-dependent cellular cytotoxicity(ADCC).

Human NK cells are controlled by positive and negative cytolyticsignals. Negative (inhibitory) signals are transduced by C-lectin domaincontaining receptors CD94/NKG2A and by some Killer Immunoglobulin-likeReceptors (KIRs). The regulation of NK lysis by inhibitory signals isknown as the “missing self” hypothesis in which specific HLA-class Ialleles expressed on the target cell surface ligate inhibitory receptorson NK cells. The down-regulation of HLA molecules on tumor cells andsome virally infected cells (e.g. CMV) lowers this inhibition below atarget threshold and the target cells may become susceptible to NKcell-mediated lysis if the target cells also carry NK-priming andactivating molecules. TLR7, TLR8 or TLR9 agonists can activate both mDCand pDCs to produce type I IFNs and express costimulatory molecules suchas GITR-ligand, which subsequently activate NK cells to produce IFN-gand potently promote NK cell killing function.

Inhibitory receptors fall into two groups, those of the Ig-superfamilycalled Killer Immunoglobulin-like Receptors (KIRs) and those of thelectin family, the NKG2, which form dimers with CD94 at the cellsurface. KIRs have a 2- or 3-domain extracellular structure and bind toHLA-A, -B or -C. The NKG2/CD94 complexes ligate HLA-E.

Inhibitory KIRs have up to 4 intracellular domains which contain ITIMsand the best characterized are KIR2DL1, KIR2DL2 and KIR2DL3 which areknown to bind HLA-C molecules. KIR2DL2 and KIR2DL3 bind the group 1HLA-C alleles while KIR2DL1 binds to group 2 alleles. Certainleukemia/lymphoma cells express both group 1 and 2 HLA-C alleles and areknown to be resistant to NK-mediated cell lysis

With regards to positive activating signals, ADCC is thought to bemediated via CD16, and a number of triggering receptors responsible fornatural cytotoxicity have been identified, including CD2, CD38, CD69,NKRP-I, CD40, B7-2, NK-TR, NKp46, NKp30 and NKp44. In addition, severalKIR molecules with short intracytoplasmic tails are also stimulatory.These KIRs (KIR2DS1, KIR2DS2 and KIR2DS4) are known to bind to HLA-C;their extracellular domains being identical to their related inhibitoryKIRs. The activatory KIRs lack the ITIMs and instead associate with DAP12 leading to NK cell activation. The mechanism of control of expressionof inhibitory versus activatory KIRs remains unknown.

Tumor Cells

In some embodiments, the activating moiety is capable of activatingtumor cells. Several reports have described the expression of TLRs inmouse or human cancer or cancer cell lines. For example, TLR1 to TLR6are expressed by colon, lung, prostate, and melanoma mouse tumor celllines (Huang B, et al. Toll-like receptors on tumor cells facilitateevasion of immune surveillance. Cancer Res. 2005; 65(12):5009-5014.),TLR3 is expressed in human breast cancer cells (Salaun B, Coste I,Rissoan M C, Lebecque S J, Renno T. TLR3 can directly trigger apoptosisin human cancer cells. J Immunol. 2006; 176(8):4894-4901.),hepatocarcinoma and gastric carcinoma cells express TLR2 and TLR4 (HuangB, et al. Listeria monocytogenes promotes tumor growth via tumor celltoll-like receptor 2 signaling. Cancer Res. 2007; 67(9):4346-4352), andTLR9 (Droemann D, et al. Human lung cancer cells express functionallyactive Toll-like receptor 9. Respir Res. 2005; 6:1.) and TLR4 (He W, LiuQ, Wang L, Chen W, Li N, Cao X. TLR4 signaling promotes immune escape ofhuman lung cancer cells by inducing immunosuppressive cytokines andapoptosis resistance. Mol Immunol 2007; 44(11):2850-2859.) are expressedby human lung cancer cells. TLR7 and TLR8 are found in tumor cells ofhuman lung cancer (Cherfils-Vicini J, Platonova S, Gillard M, Laurans L,Validire P, Caliandro R, Magdeleinat P, Mami-Chouaib F, Dieu-Nosjean MC, Fridman W H, Damotte D, Sautes-Fridman C, Cremer I. J. Clin Invest.2010; 120(4):1285-1297).

Effect on tumor cells could be various, for instance, stimulation ofTLR3 by poly I:C in human breast cancer and melanoma cells directlytriggers apoptosis of tumor cells (Salaun B, Coste I, Rissoan M C,Lebecque S J, Renno T. TLR3 can directly trigger apoptosis in humancancer cells. J Immunol. 2006; 176(8):4894-4901., Salaun B, Lebecque S,Matikainen S, Rimoldi D, Romero P. Toll-like receptor 3 expressed bymelanoma cells as a target for therapy? Clin Cancer Res. 2007; 13(15 Pt1):4565-4574.). On the other hand, Human lung cancer cells expressfunctionally active Toll-like receptor 9 and stimulation of the TLR-9expressing cell lines A549 and HeLa with CpG-ODN showed anti apoptoticeffect. Nevertheless, accumulated data shows TLRs are well associatedwith cancer cells and may effect on it directly or indirectly.

Chemical Composition of Activating Moiety

In general, activating moiety of the present invention comprises aprotein, an antibody, a nucleic acid, a small molecule, or a ligand.

In some embodiments, the AM is capable of binding specifically to humanTLR7 and/or TLR8.

In some embodiments, the activating moiety comprises: (a)single-stranded RNA (ssRNA), preferably ORN02, ORN06, ssPoly(U),ssRNA40, ssRNA41, ssRNA-DR, or Poly(dT); or (b) a ligand analogs,preferably CL075, CL097, CL264, CL307, Gardiquimod, Loxoribine,Imiquimod, or Resiquimod.

In some embodiments, the activating moiety comprises a TLR ligand(TLRL), a Nod-Like Receptor ligand, a RIG-Like Receptor ligand, a CLRligand, a CDS ligand, or a inflammasome inducers.

In some embodiments, the activating moiety comprises a ligand of one ormore TLRs selected from the group consisting of: TLR2, TLR3, TLR4, TLR5,TLR7, TLR8, TLR7/TLR8, TLR9 and TLR10.

TLR are a family of proteins that sense a microbial product and/orinitiates an adaptive immune response. TLRs activate a dendritic cell(DC). TLRs are conserved membrane spanning molecules containing anectodomain of leucine-rich repeats, a transmembrane domain and anintracellular TIR (Toll/interleukin receptor) domain. TLRs recognizedistinct structures in microbes, often referred to as “PAMPs” (pathogenassociated molecular patterns). Ligand binding to TLRs invokes a cascadeof intracellular signaling pathways that induce the production offactors involved in inflammation and immunity.

Exemplary agonists which may be used for these receptors include,without limitation lipoproteins, lipopolypeptides, peptidoglycans,zymosan, lipopolysaccharide, neisserial porins, flageilin, profiilin,galactoceramide, muramyl dipeptide, giucopyranosyl lipid A (GLA), andresiquimod (R848). Peptidoglycans, lipoproteins, and lipoteichoic acidsare cell wall components of Gram-positive. Lipopolysaccharides areexpressed by most bacteria. Flageilin is the structural component ofbacterial flagella that is secreted by pathogenic and commensalbacterial. A Galactosylceramide (α-GalCer) is an activator of naturalkiller T (NKT) cells. Muramyl dipeptide is a bioactive peptidoglycanmotif common to all bacteria. Such agonists mediate innate immuneactivation via Toll-like Receptors. Specific binding of an agonist forits cognate receptor is often expressed in terms of an affinity. Theligands of the present invention may bind with affinities of betweenabout 10⁴M⁻¹ and about 10⁸ M⁻¹. Affinity is calculated asK_(d)=k_(off)/k_(on), (k_(off) is the dissociation rate constant, K_(on)is the association rate constant and K_(d) is the equilibrium constant).Single or multiple agonists may be used.

In humans, at least eight TLRs have been identified. TLRs that areexpressed on the surface of cells include TLR2, TLR3, TLR4, TLR5, TLR7,TLR8 or TLR7/TLR8, TLR9 and TLR10 are expressed with the ER compartment.Human dendritic cell subsets can be identified on the basis of distinctTLR expression patterns. By way of example, the myeloid or“conventional” subset of DC (mDC) expresses TLRs 2-8 when stimulated,and a cascade of activation markers (e.g. CD80, CD86, MHC class I andII, CCR7), pro-inflammatory cytokines, and chemokmes are produced. Aresult of this stimulation and resulting expression is antigen-specificCD4+ and CD8+ T cell priming. These DCs acquire an enhanced capacity totake up antigens and present them in an appropriate form to T cells. Incontrast, the plasmacytoid subset of DC (pDC) expresses only TLR7 andTLR9 upon activation, with a resulting activation of NK cells as well asT-cells. Without being bound by any particular theory, as dying tumorcells may adversely affect DC function, it is hypothesized thatactivating DC with TLR agonists is beneficial for priming anti-tumorimmunity in an immunotherapy approach to the treatment of cancer. It hasalso been suggested that successful treatment of breast cancer usingradiation and chemotherapy requires TLR4 activation.

TLR agonists known in the art and useful in the present inventioninclude, but are not limited to, the following: Pam3Cys, a TLR-1/2agonist; cell-wall components products (LAM, LM, LPS, LTA, LTA), a TLR-2agonist; MALP2, a TLR-2 agonist; Pam2Cys, a TLR-2 agonist; FSL, a TLR-2agonist; Zymosan, a TLR-2 agonist; PGN, a TLR-2 agonist; polyribosinic;polyribocytidic acid (Poly I:C), a TLR-3 agonist;polyadenosine-polyuridylie acid (poly AU), a TLR-3 agonist;Polyinosinie-Polycytidylic acid stabilized with poiy-L-lysine andcarboxymethylcelitilose (Hiltonol©), a TLR-3 agonist; monophosphoryllipid A (MPL), a TLR-4 agonist; LPS, a TLR-4 agonist; bacterialflagellin, a TLR-5 agonist; FLA, a TLR-5 agonist; ORN02, a TLR-7/8agonist; ORN06, a TLR-7/8 agonist; ssPoly(U), a TLR-7/8 agonist;ssRNA40, a TLR-7/8 agonist; ssRNA41, a TLR-7/8 agonist; ssRNA-DR, a 7/8agonist; Poly(dT), a TLR-7/8 agonist; CL075, a TLR-7/8 agonist; CL097, aTLR-7/8 agonist; CL264, a TLR-7/8 agonist; CL307, a TLR-7/8 agonist;Gardiquimod, a TLR-7/8 agonist; Loxoribine, a TLR-7/8 agonist;Imiquimod, a TLR-7/8 agonist; Resiquimod a TLR-7/8 agonist; and ODN1585,a TLR-9 agonist; ODN1668, a TLR-9 agonist; ODN1826, a TLR-9 agonist;ODN2006, a TLR-9 agonist; ODN2007, a TLR-9 agonist; ODN2216, a TLR-9agonist; ODN2336, a TLR-9 agonist; ODN2395, a TLR-9 agonist; ODN M362 aTLR-9 agonist.

In some embodiments, the activating moiety comprises:

(i) a ligand of TLR2 selected from the group consisting of: (a) a heatkilled bacteria product, preferably HKAL, HKEB, HKHP, HKLM, HKLP, HKLR,HKMF, HKPA, HKPG, or HKSA, HKSP, and (b) a cell-wall components product,preferably LAM, LM, LPS, LTA, LTA, PGN, FSL, Pam2CSK4, Pam3CSK4, orZymosan;

(ii) a ligand of TLR3 selected from the group consisting of: Poly (I:C)and Poly (A:U);

(iii) a ligand of TLR4 selected from the group consisting of: LPS andMPLA;

(iv) a ligand of TLR5 selected from the group consisting of: FLA andFlagellin;

(v) a ligand of TLR7, TLR 8, or TLR 7 and TLR, selected from the groupconsisting of: ORN02 (TLR8), ORN06 (TLR8), ssPoly(U) (TLR8), ssRNA40(TLR8), ssRNA41 (TLR8), ssRNA-DR (TLR8) Poly(dT) (TLR7/8), CL075(TLR7/8), CL097 (TLR7/8), CL264 (TLR7), CL307 (TLR7), Gardiquimod(TLR7), Loxoribine (TLR7), Imiquimod (TLR7/8), and Resiquimod (TLR7/8);

(vi) a ligand of TLR9 selected from the group consisting of: ODN1585,ODN1668, ODN1826, ODN2006, ODN2007, ODN2216, ODN2336, ODN2395, and ODNM362;

(vii) a ligand of TLR10;

(viii) a ligand of nucleotide-oligomerization domain (NOD)-like selectedfrom the group consisting of: NOD1 agonist (C12-iE-DAP, iE-DAP,Tri-DAP), NOD2 agonist (L18-MDP, MDP, M-TriLYS, M-TriLYS-D-ASN,Murabutide, N-Glycolyl-MDP), and NOD1/NOD2 agonists (M-TriDAP, PGN);

(ix) a ligand of one or more RIG-I-Like receptors (RLR) selected fromthe group consisting of: 5′ppp-dsRNA, Poly (dA:dT), Poly(dG:dC), andPoly (I:C);

(x) a ligand of one or more C-type lectin receptors (CLR) selected fromthe group consisting of: Curdlan AL, HKCA, HKSC, WGP, Zymosan, andTrehalose-6,6-dibehenate;

(xi) a ligand of one or more Cytosolic DNA Sensors (CDS) selected fromthe group consisting of: c-GMP, c-G-AMP, c-G-GMP, c-A-AMP, c-di-AMP,c-di-IMP, c-di-GMP, c-di-UMP, HSV-60, ISD, pCpG, Poly (dA:dT),Poly(dG:dC), Poly (dA), and VACV-70; and

(xii) an inflammasome inducer selected from the group consisting of: (a)NLRP3 inflammasome protein complex, preferably alum Crystals, ATP, CPPDCrystals, Hermozoin, MSU Crystals, Nano-SiO2, Nigericin, and (b) AIM2inflammasome protein complex, preferably Poly (dA:dT).

In some embodiments, the activating moiety comprises IMO2134, IMO205,MGN-1703, MGN-1704, Agatolimod, SD-101, QAX-935, DIMS0150, PollinexQuattro, OM-174, Eritoran, TAK-242, NI-0101, Type I interferon, type IIinterferon, type III interferon, IL-12, IL-23, IL-18, IL-7 or IL-15.

In some embodiments, the activating moiety is capable of bindingspecifically to human TLR7 and/or TLR8, preferably binds specifically toboth TLR7 and TLR8, such as Resiquimod (R848), thus is capable ofactivating both pDC and mDC. TLR7 and TLR8 are phylogenetically andstructurally related. TLR7 is selectively expressed by human pDCs and Bcells. TLR8 is predominantly expressed mDCs, monocytes, macrophages andmyeloid suppressor cells. TLR7-specific agonists activate plasmacytoidDCs (pDCs) to produce large amounts of type 1 IFNs and expressing highlevels of costimulatory molecules that promote activation of T cells, NKcells, B cells and mDCs. TLR8-specific agonists activate myeloid DCs,monocytes, macrophages or myeloid-derived suppressor cells to producelarge amounts of type 1 IFN, IL-12 and IL-23, and express high levels ofMHC class I, MHC class II and costimulatory molecules that promote theactivation of antigen specific CD4 and CD8+ T cells.

Additional moieties or molecules that activate human immune cellsthrough TLR can be identified by TLR reporter cell lines, e.g.: 293/TLRreporter cells from InvivoGen.

Additional moieties or molecules that can activate TLR7 and/or TLR 8 canbe identified using using the 293/TLR reporter cells from InvivoGen, andthe ability of these additional moieties to stimulate pDCs to producetype 1 IFN, and to stimulate mDCs to produce type 1 IFN and IL-12, aswell as to upregulate the expression of CD80, CD86.

The activating moiety that is capable of activating NK cells via type 1IFN and co-stimulatory molecules such as GITR-ligand expressed byactivated pDC and/or mDC can be identified by NK cell proliferation andexpression of activation maker CD69 in total peripheral bloodmononuclear cell assay.

The activating moiety that is capable of activating tumor cells toundergo cell death and to produce cytokines such as type 1 IFN, IL-1a/b,TNF or IL-6 can be identified using methods known in the art.

Additional activating moieties or molecules that are capable ofactivating DC cells, monocytes, macrophages, myeloid suppressor cells, Bcells, NK cells, or tumor cells can be identified using methods known inthe art.

In some embodiments, when the targeting moiety comprises an antibody (ora fragment thereof), or an aptamer, the activating moiety does notcomprise a CpG oligonucleotide.

In some embodiments, specially when the targeting moiety comprises anantibody or a fragment there of, or an aptamer, the activating moietydoes not comprise a therapeutic agent, such as chemotherapeutic agentsor toxins, such as toxins generally used in antibody-drug conjugates).Thus, in general, the compounds of the present invention do notencompass antibody-drug conjugates (ADC), or antibody-CpG conjugates.

C. Linkers

In general, the compound of the invention comprises a linker that linksthe targeting moiety and the activating moiety. Though, in some compoundthere is no linker and the activating moiety and the targeting moiety islinked directly.

By “linker” herein is meant a moiety that connects a first molecule to asecond molecule through chemical bonds. In linkers of the invention, theconnection can be severed so as to release a biologically active form ofthe first and/or second molecule. A preferred example of a linker is amoiety that comprises a bond that is stable at neutral pH but is readilycleaved under conditions of low pH. Particularly preferred examples oflinkers are moieties that comprise a bond that is stable at pH valuesbetween 7 and 8 but is readily cleaved at pH values between 4 and 6.Another example of a linker is a moiety that comprises a bond that isreadily cleaved in the presence of an enzyme. Preferred examples of suchenzyme-sensitive linkers are peptides comprising a recognition sequencefor an endosomal peptidase. Another example of a linker is a redoxpotential-sensitive linker that is stable under conditions of lowreduction potential (e.g., low thiol or glutathione concentration) butcleaved under conditions of high reduction potential (e.g., high thiolor glutathione concentration). Preferred examples of such redoxpotential-sensitive linkers include disulfides and sulfenamides.Particularly preferred examples include substituted aryl-alkyldisulfides in which the aryl group is substituted withsterically-demanding and electron-withdrawing or electron-donatingsubstitutents, so as to control the sensitivity of the disulfide linkagetowards reaction with thiol. Another example of a linker is a moietythat comprises a bond that is readily cleaved upon exposure toradiation. Preferred examples of such radiation-sensitive linkers are2-nitrobenzyl ethers that are cleaved upon exposure to light.Particularly preferred examples of linkers are moieties that mask thebiological activity of one of the two linked molecules until the linkageis severed.

In some embodiments, the compound of the invention comprises a linkerthat is selected from the group consisting of a hydrazine group, apolypeptide, a disulfide group, and a thioether group.

By “hydrazine group” or “hydrazine linker” or “self-cyclizing hydrazinelinker” herein is meant a linker moiety that, upon a change incondition, such as a shift in pH, will undergo a cyclization reactionand form one or more rings. The hydrazine moiety is converted to ahydrazone when attached. This attachment can occur, for example, througha reaction with a ketone group on the L4 moiety. Therefore, the termhydrazine linker can also be used to describe the linker of the currentinvention because of this conversion to a hydrazone upon attachment.

By “five-membered hydrazine linker” or “5-membered hydrazine linker”herein is meant hydrazine-containing molecular moieties that, upon achange in condition, such as a shift in pH, will undergo a cyclizationreaction and form one or more 5-membered rings. Alternatively, this fivemembered linker may similarly be described as a five-membered hydrazinelinker or a 5-membered hydrazine linker.

By “six-membered hydrazine linker” or “6-membered hydrazine linker”herein is meant hydrazine-containing molecular moieties that, upon achange in condition such as a shift in pH, will undergo a cyclizationreaction and form one or more 6-membered rings. This six membered linkermay similarly be described as a six-membered hydrazine linker or a6-membered hydrazine linker.

By “cyclization reaction” herein is meant the cyclization of a peptide,hydrazine, or disulfide linker, indicates the cyclization of that linkerinto a ring and initiates the separation of the drug-ligand complex.This rate can be measured ex situ, and is completed when at least 90%,95%, or 100% of the product is formed.

In some embodiments, the compound of the present invention comprises alinker region between the targeting moiety and the activating moiety,and the linker is cleavable by a cleaving agent that is present in theintracellular environment (e.g., within a lysosome or endosome orcaveolea). The linker can be, e.g., a peptidyl linker that is cleaved byan intracellular peptidase or protease enzyme, including, but notlimited to, a lysosomal or endosomal protease. Typically, the peptidyllinker is at least two amino acids long or at least three amino acidslong. Cleaving agents can include cathepsins B and D and plasmin, all ofwhich are known to hydrolyze dipeptide drug derivatives resulting in therelease of active drug inside target cells (see, e.g., Dubowchik andWalker, 1999, Pharm. Therapeutics 83:67-123). Most typical are peptidyllinkers that are cleavable by enzymes that are present in targeted cellsor tissues. For example, a peptidyl linker that is cleavable by thethiol-dependent protease cathepsin-B, which is highly expressed incancerous tissue, can be used (e.g., a Phe-Leu or a Gly-Phe-Leu-Gly)linker). Other such linkers are described, e.g., in U.S. Pat. No.6,214,345. In some embodiments, the peptidyl linker cleavable by anintracellular protease is a Val-Cit linker or a Phe-Lys linker (see,e.g., U.S. Pat. No. 6,214,345, which describes the synthesis ofdoxorubicin with the val-cit linker). One advantage of usingintracellular proteolytic release of the therapeutic agent is that theagent is typically attenuated when conjugated and the serum stabilitiesof the conjugates are typically high.

In some embodiments, the cleavable linker is pH-sensitive, i.e.,sensitive to hydrolysis at certain pH values. Typically, thepH-sensitive linker hydrolyzable under acidic conditions. For example,an acid-labile linker that is hydrolyzable in the lysosome (e.g., ahydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide,orthoester, acetal, ketal, or the like) can be used. (See, e.g., U.S.Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999,Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem.264:14653-14661.) Such linkers are relatively stable under neutral pHconditions, such as those in the blood, but are unstable at below pH 5.5or 5.0, the approximate pH of the lysosome. In certain embodiments, thehydrolyzable linker is a thioether linker (such as, e.g., a thioetherattached to the therapeutic agent via an acylhydrazone bond (see, e.g.,U.S. Pat. No. 5,622,929)).

In yet other embodiments, the linker is cleavable under reducingconditions (e.g., a disulfide linker). A variety of disulfide linkersare known in the art, including, for example, those that can be formedusing SATA (N-succinimidyl-5-acetylthioacetate), SPDP(N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB(N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT(N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene),SPDB and SMPT (See, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931;Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates inRadioimagery and Therapy of Cancer (C. W. Vogel ed., Oxford U. Press,1987. See also U.S. Pat. No. 4,880,935.)

In yet other specific embodiments, the linker is a malonate linker(Johnson et al., 1995, Anticancer Res. 15:1387-93), a maleimidobenzoyllinker (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a3′-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12).

Typically, the linker is not substantially sensitive to theextracellular environment. As used herein, “not substantially sensitiveto the extracellular environment,” in the context of a linker, meansthat no more than about 20%, typically no more than about 15%, moretypically no more than about 10%, and even more typically no more thanabout 5%, no more than about 3%, or no more than about 1% of thelinkers, in a sample of compounds of the present invention, are cleavedwhen the compounds of the present invention present in an extracellularenvironment (e.g., in plasma). Whether a linker is not substantiallysensitive to the extracellular environment can be determined, forexample, by incubating independently with plasma both (a) the compoundof the invention (the “Compound sample”) and (b) an equal molar amountof unconjugated antibody or therapeutic agent (the “control sample”) fora predetermined time period (e.g., 2, 4, 8, 16, or 24 hours) and thencomparing the amount of unconjugated antibody or therapeutic agentpresent in the Compound sample with that present in control sample, asmeasured, for example, by high performance liquid chromatography.

In other, non-mutually exclusive embodiments, the linker promotescellular internalization. In certain embodiments, the linker promotescellular internalization when conjugated to the activating moiety. Inyet other embodiments, the linker promotes cellular internalization whenconjugated to both the targeting moiety and the activating moiety.

A variety of linkers that can be used with the present compositions andmethods are described in WO 2004010957 entitled “Drug Conjugates andTheir Use for Treating Cancer, An Autoimmune Disease or an InfectiousDisease”, US20120141509A1, and US20120288512A1 (the disclosure of whichis incorporated by reference herein).

In certain embodiments, the linker unit has the following generalformula:

-Ta-Ww-Yy-

wherein -T- is a stretcher unit; a is 0 or 1; each —W— is independentlyan amino acid unit; w is independently an integer ranging from 2 to 12;—Y— is a spacer unit; and y is 0, 1 or 2.

The Stretcher Unit

The stretcher unit (-T-), when present, links the targeting moiety to anamino acid unit (—W—). Useful functional groups that can be present on atargeting moiety, such as an antibody, either naturally or via chemicalmanipulation include, but are not limited to, sulthydryl, amino,hydroxyl, the anomeric hydroxyl group of a carbohydrate, and carboxyl.Suitable functional groups are sulthydryl and amino. Sulthydryl groupscan be generated by reduction of the intramolecular disulfide bonds ofan antibody. Alternatively, sulthydryl groups can be generated byreaction of an amino group of a lysine moiety of an antibody with2-iminothiolane (Traut's reagent) or other sulthydryl generatingreagents. In some embodiments, the antibody is a recombinant antibodyand is engineered to carry one or more lysines. In other embodiments,the recombinant antibody is engineered to carry additional sulthydrylgroups, e.g., additional cysteines.

In some embodiments, the stretcher unit forms a bond with a sulfur atomof the antibody. The sulfur atom can be derived from a sulthydryl (—SH)group of a reduced antibody (A). Representative stretcher units of theseembodiments are depicted within the square brackets of Formulas (IIa)and (IIb), wherein A-, —W—, —Y—, -D, w and y are as defined above and R¹is selected from —C₁-C₁₀ alkylene-, —C₃-C₈ carbocyclo-, —O—(C₁-C₈alkyl)-, -arylene-, —C₁-C₁₀ alkylene-arylene-, -arylene-C₁-C₁₀alkylene-, —C₁-C₁₀ alkylene-(C₃-C₈ carbocyclo)-, —(C₃-C₈carbocyclo)-C₁-C₁₀alkylene-, —C₃-C₈ heterocyclo-, —C₁-C₁₀alkylene-(C₃-C₈ heterocyclo)-, —(C₃-C₈ heterocyclo)-C₁-C₁₀ alkylene-,—(CH₂CH₂O)r-, and —(CH₂CH₂O)r-CH₂—; and r is an integer ranging from1-10.

An illustrative stretcher unit is that of formula (IIa) where R¹ is—(CH₂)₅—:

Another illustrative stretcher unit is that of formula (IIa) where R¹ is—(CH₂CH₂O)r-CH₂— and r is 2:

Still another illustrative stretcher unit is that of formula (IIb) whereR¹ is —(CH₂)₅—:

In certain other specific embodiments, the stretcher unit is linked tothe antibody unit (A) via a disulfide bond between a sulfur atom of theantibody unit and a sulfur atom of the stretcher unit. A representativestretcher unit of this embodiment is depicted within the square bracketsof Formula (III), wherein R¹, A-, —W—, —Y—, -D, w and y are as definedabove.

AS—R¹—C(O)W_(w)—Y_(y)-D  (III)

In other specific embodiments, the reactive group of the stretchercontains a reactive site that can be reactive to an amino group of anantibody. The amino group can be that of an arginine or a lysine.Suitable amine reactive sites include, but are not limited to, activatedesters such as succinimide esters, 4-nitrophenyl esters,pentafluorophenyl esters, anhydrides, acid chlorides, sulfonylchlorides, isocyanates and isothiocyanates. Representative stretcherunits of these embodiments are depicted within the square brackets ofFormulas (IVa) and (IVb), wherein R¹, A-, —W—, —Y—, -D, w and y are asdefined above;

In yet another aspect, the reactive function of the stretcher contains areactive site that is reactive to a modified carbohydrate group that canbe present on an antibody. In some embodiments, the antibody isglycosylated enzymatically to provide a carbohydrate moiety. Thecarbohydrate may be mildly oxidized with a reagent such as sodiumperiodate and the resulting carbonyl unit of the oxidized carbohydratecan be condensed with a stretcher that contains a functionality such asa hydrazide, an oxime, a reactive amine, a hydrazine, athiosemicarbazide, a hydrazine carboxylate, and an arylhydrazide such asthose described by Kaneko et al., 1991, Bioconjugate Chem 2:133-41.Representative stretcher units of this embodiment are depicted withinthe square brackets of Formulas (Va)-(IVc), wherein R¹, A-, —W—, —Y—,-D, w and y are as defined above.

The Amino Acid Unit

The amino acid unit (—W—) links the stretcher unit (-T-) to the Spacerunit (—Y—) if the Spacer unit is present, and links the stretcher unitto the cytotoxic or cytostatic agent (Activating Moiety; D) if thespacer unit is absent. —Ww- is a dipeptide, tripeptide, tetrapeptide,pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide,decapeptide, undecapeptide or dodecapeptide unit. Each —W— unitindependently has the formula denoted below in the square brackets, andw is an integer ranging from 2 to 12:

wherein R² is hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl,p-hydroxybenzyl, —CH₂OH, —CH(OH)CH₃, —CH₂CH₂SCH₃, —CH₂CONH₂, —CH₂COOH,—CH₂CH₂CONH₂, —CH₂CH₂COOH, —(CH₂)₃NHC(═NH)NH₂, —(CH₂)₃NH₂,—(CH₂)₃NHCOCH₃, —(CH₂)₃NHCHO, —(CH₂)₄NHC(═NH)NH₂, —(CH₂)₄NH₂,—(CH₂)₄NHCOCH₃, —(CH₂)₄NHCHO, —(CH₂)₃NHCONH₂, —(CH₂)₄NHCONH₂,—CH₂CH₂CH(OH)CH₂NH₂, 2-pyridylmethyl-, 3-pyridylmethyl-,4-pyridylmethyl-, phenyl, cyclohexyl,

The amino acid unit of the linker unit can be enzymatically cleaved byan enzyme including, but not limited to, a tumor-associated protease toliberate the activating moiety (-D) which is protonated in vivo uponrelease to provide an activating molecule (D).

Illustrative W_(w) units are represented by formulas (VI)-(VIII):

wherein R³ and R⁴ are as follows:

R³ R⁴ Benzyl (CH₂)₄NH₂; Methyl (CH₂)₄NH₂; Isopropyl (CH₂)₄NH₂; Isopropyl(CH₂)₃NHCONH₂; Benzyl (CH₂)₃NHCONH₂; Isobutyl (CH₂)₃NHCONH₂; sec-butyl(CH₂)₃NHCONH₂;

(CH₂)₃NHCONH₂; Benzyl methyl; and Benzyl (CH₂)₃NHC(═NH)NH₂;

wherein R³, R⁴ and R⁵ are as follows:

R³ R⁴ R⁵ Benzyl benzyl (CH₂)₄NH₂; Isopropyl benzyl (CH₂)₄NH₂; and Hbenzyl (CH₂)₄NH₂;

wherein R³, R⁴, R⁵ and R⁶ are as follows:

R³ R⁴ R⁵ R⁶ H Benzyl isobutyl H; and methyl Isobutyl methyl isobutyl.

Suitable amino acid units include, but are not limited to, units offormula (VI) where: R³ is benzyl and R⁴ is —(CH₂)₄NH₂; R₃ is isopropyland R⁴ is —(CH₂)₄NH₂; or R³ is isopropyl and R⁴ is —(CH₂)₃NHCONH₂.Another suitable amino acid unit is a unit of formula (VII), where: R³is benzyl, R⁴ is benzyl, and R⁵ is —(CH₂)₄NH₂. —Ww-units can be designedand optimized in their selectivity for enzymatic cleavage by aparticular tumor-associated protease. The suitable —Ww- units are thosewhose cleavage is catalyzed by the proteases, cathepsin B, C and D, andplasmin.

In some embodiments, —Ww- is a dipeptide, tripeptide or tetrapeptideunit.

Where R², R³, R⁴, R⁵ or R⁶ is other than hydrogen, the carbon atom towhich R², R³, R⁴, R⁵ or R⁶ is attached is chiral. Each carbon atom towhich R², R³, R⁴, R⁵ or R⁶ is attached is independently in the (S) or(R) configuration.

In some embodiments, the amino acid unit is a phenylalanine-lysinedipeptide (Phe-Lys or FK linker). In some embodiments, the amino acidunit is a valine-citrulline dipeptide (Val-Cit or VC linker). In someembodiments, the amino acid unit is 5-aminovaleric acid, homophenylalanine lysine, tetraisoquinolinecarboxylate lysine,cyclohexylalanine lysine, isonepecotic acid lysine, beta-alanine lysine,glycine serine valine glutamine, or isonepecotic acid.

The amino acid unit can comprise natural amino acids. In otherembodiments, the Amino Acid unit can comprise non-natural amino acids.

The Spacer Unit

The spacer unit (—Y—), when present, links an amino acid unit to thedrug unit. Spacer units are of two general types: self-immolative andnon self-immolative. A non self-immolative spacer unit is one in whichpart or all of the spacer unit remains bound to the activating moietyunit after enzymatic cleavage of an amino acid unit from theTM-linker-AM conjugate or the drug-linker compound. Examples of a nonself-immolative spacer unit include, but are not limited to a(glycine-glycine) spacer unit and a glycine spacer unit. When aTM-linker-AM conjugate containing a glycine-glycine spacer unit or aglycine spacer unit undergoes enzymatic cleavage via a tumor-cellassociated-protease, a cancer-cell-associated protease or alymphocyte-associated protease, a glycine-glycine-drug moiety or aglycine-drug moiety is cleaved from A-T-Ww-. To liberate the AM, anindependent hydrolysis reaction should take place within the target cellto cleave the glycine-drug unit bond.

In a typical embodiment, -Yy- is a p-aminobenzyl ether which can besubstituted with Qm where Q is —C₁-C₈ alkyl, —C1-C₈alkoxy, -halogen,-nitro or -cyano; and m is an integer ranging from 0-4.

In some embodiments, a non self-immolative spacer unit (—Y—) is-Gly-Gly-.

In some embodiments, a non self-immolative the spacer unit (—Y—) is-Gly-.

In one embodiment, the AM-linker compound or an TM-linker-AM conjugatelacks a spacer unit (y=0).

Alternatively, an TM-linker-AM conjugate containing a self-immolativespacer unit can release the AM (D) without the need for a separatehydrolysis step. In these embodiments, —Y— is a p-aminobenzyl alcohol(PAB) unit that is linked to —Ww- via the nitrogen atom of the PABgroup, and connected directly to -D via a carbonate, carbamate or ethergroup.

Other examples of self-immolative spacers include, but are not limitedto, aromatic compounds that are electronically equivalent to the PABgroup such as 2-aminoimidazol-5-methanol derivatives (see Hay et al.,1999, Bioorg. Med. Chem. Lett. 9:2237 for examples) and ortho orpara-aminobenzylacetals. Spacers can be used that undergo facilecyclization upon amide bond hydrolysis, such as substituted andunsubstituted 4-aminobutyric acid amides (Rodrigues et al., 1995,Chemistry Biology 2:223), appropriately substituted bicyclo[2.2.1] andbicyclo[2.2.2] ring systems (Storm et al., 1972, J. Amer. Chem. Soc.94:5815) and 2-aminophenylpropionic acid amides (Amsberry et al., 1990,J. Org. Chem. 55:5867) Elimination of amine-containing drugs that aresubstituted at the α-position of glycine (Kingsbury, et al., 1984, J.Med. Chem. 27:1447) are also examples of self-immolative spacerstrategies that can be applied to the TM-linker-AM conjugates.

In an alternate embodiment, the spacer unit is a branchedbis(hydroxymethyl)styrene (BHMS) unit, which can be used to incorporatemoieties.

Typical spacer units (—Yy-) are represented by Formulas (IX)-(XI):

where Q is C₁-C₈ alkyl, C₁-C₈ alkoxy, halogen, nitro or cyano; and m isan integer ranging from 0-4;

In some embodiments, the linker is enzymatic cleavable. In someembodiments, the linker is not enzymatic cleavable.

III. PHARMACEUTICAL FORMULATIONS AND ADMINISTRATION

The present invention further relates to a pharmaceutical formulationcomprising a compound of the invention or a pharmaceutically acceptablesalt thereof, and one or more pharmaceutically acceptable carriers.

The compounds described herein including pharmaceutically acceptablecarriers such as addition salts or hydrates thereof, can be delivered toa patient using a wide variety of routes or modes of administration.Suitable routes of administration include, but inhalation, transdermal,oral, rectal, transmucosal, intestinal and parenteral administration,including intramuscular, subcutaneous and intravenous injections.Preferably, the compounds of the invention comprising an antibody orantibody fragment as the targeting moiety are administered parenterally,more preferably intravenously.

As used herein, the terms “administering” or “administration” areintended to encompass all means for directly and indirectly delivering acompound to its intended site of action.

The compounds described herein, or pharmaceutically acceptable saltsand/or hydrates thereof, may be administered singly, in combination withother compounds of the invention, and/or in cocktails combined withother therapeutic agents. Of course, the choice of therapeutic agentsthat can be co-administered with the compounds of the invention willdepend, in part, on the condition being treated.

For example, when administered to patients suffering from a diseasestate caused by an organism that relies on an autoinducer, the compoundsof the invention can be administered in cocktails containing agents usedto treat the pain, infection and other symptoms and side effectscommonly associated with the disease. Such agents include, e.g.,analgesics, antibiotics, etc.

When administered to a patient undergoing cancer treatment, thecompounds may be administered in cocktails containing anti-cancer agentsand/or supplementary potentiating agents. The compounds may also beadministered in cocktails containing agents that treat the side-effectsof radiation therapy, such as anti-emetics, radiation protectants, etc.

Supplementary potentiating agents that can be co-administered with thecompounds of the invention include, e.g., tricyclic anti-depressantdrugs (e.g., imipramine, desipramine, amitriptyline, clomipramine,trimipramine, doxepin, nortriptyline, protriptyline, amoxapine andmaprotiline); non-tricyclic and anti-depressant drugs (e.g., sertraline,trazodone and citalopram); Ca+2 antagonists (e.g., verapamil,nifedipine, nitrendipine and caroverine); amphotericin; triparanolanalogues (e.g., tamoxifen); antiarrhythmic drugs (e.g., quinidine);antihypertensive drugs (e.g., reserpine); thiol depleters (e.g.,buthionine and sulfoximine); and calcium leucovorin.

The active compound(s) of the invention are administered per se or inthe form of a pharmaceutical composition wherein the active compound(s)is in admixture with one or more pharmaceutically acceptable carriers,excipients or diluents. Pharmaceutical compositions for use inaccordance with the present invention are typically formulated in aconventional manner using one or more physiologically acceptablecarriers comprising excipients and auxiliaries, which facilitateprocessing of the active compounds into preparations which, can be usedpharmaceutically. Proper formulation is dependent upon the route ofadministration chosen.

For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art.

For oral administration, the compounds can be formulated readily bycombining the active compound(s) with pharmaceutically acceptablecarriers well known in the art. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, and suspensions for oral ingestion by apatient to be treated. Pharmaceutical preparations for oral use can beobtained solid excipient, optionally grinding a resulting mixture, andprocessing the mixture of granules, after adding suitable auxiliaries,if desired to obtain tablets or dragee cores. Suitable excipients are,in particular, fillers such as sugars, including lactose, sucrose,mannitol, or sorbitol; cellulose preparations such as, for example,maize starch, wheat starch, rice starch, potato starch, gelatin, gumtragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxyniethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired,disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodiumalginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations, which can be used orally, include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Injection isa preferred method of administration for the compositions of the currentinvention. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multi-dose containers, with an addedpreservative. The compositions may take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents may be added, such as the cross-linked polyvinyl pyrrolidone,agar, or alginic acid or a salt thereof such as sodium alginate.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances, which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents, which increase the solubility of thecompounds to allow for the preparation of highly, concentratedsolutions. For injection, the agents of the invention may be formulatedin aqueous solutions, preferably in physiologically compatible bufferssuch as Hanks's solution, Ringer's solution, or physiological salinebuffer.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation or transcutaneous delivery (e.g.,subcutaneously or intramuscularly), intramuscular injection or atransdermal patch. Thus, for example, the compounds may be formulatedwith suitable polymeric or hydrophobic materials (e.g., as an emulsionin an acceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude calcium carbonate, calcium phosate, various sugars, starches,cellulose derivatives, gelatin, and polymers such as polyethyleneglycols.

A preferred pharmaceutical composition is a composition formulated forinjection such as intravenous injection and includes about 0.01% toabout 100% by weight of the compound of the present invention, basedupon 100% weight of total pharmaceutical composition. The drug-ligandconjugate may be an antibody-cytotoxin conjugate where the antibody hasbeen selected to target a particular cancer.

In some embodiments, the pharmaceutical composition of the presentinvention further comprises a second chemotherapeutic agent.

By “chemotherapeutic agent” herein is meant a chemical compound usefulin the treatment of cancer. Examples are but not limited to:Gemcitabine, Irinotecan, Doxorubicin, 5-Fluorouracil, Cytosinearabinoside (“Ara-C”), Cyclophosphamide, Thiotepa, Busulfan, Cytoxin,TAXOL, Methotrexate, Cisplatin, Melphalan, Vinblastine and Carboplatin.

In some embodiments, the second chemotherapeutic agent is selected fromthe group consisting of tamoxifen, raloxifene, anastrozole, exemestane,letrozole, imatanib, paclitaxel, cyclophosphamide, lovastatin, minosine,gemcitabine, cytarabine, 5-fluorouracil, methotrexate, docetaxel,goserelin, vincristine, vinblastine, nocodazole, teniposide etoposide,gemcitabine, epothilone, vinorelbine, camptothecin, daunorubicin,actinomycin D, mitoxantrone, acridine, doxorubicin, epirubicin, oridarubicin.

IV. KITS

In another aspect, the present invention provides kits containing one ormore of the compounds or compositions of the invention and directionsfor using the compound or composition. In an exemplary embodiment, theinvention provides a kit for conjugating a linker arm of the inventionto another molecule. The kit includes the linker, and directions forattaching the linker to a particular functional group. The kit may alsoinclude one or more of a cytotoxic drug, a targeting agent, a detectablelabel, pharmaceutical salts or buffers. The kit may also include acontainer and optionally one or more vial, test tube, flask, bottle, orsyringe. Other formats for kits will be apparent to those of skill inthe art and are within the scope of the present invention.

V. MEDICAL USE

In another aspect, the present invention provides a method for treatinga disease condition in a subject that is in need of such treatment,comprising: administering to the subject a pharmaceutical compositioncomprising a therapeutically effective amount of the compound of thepresent invention a pharmaceutically acceptable salt thereof, and apharmaceutical acceptable carrier.

In addition to the compositions and constructs described above, thepresent invention also provides a number of uses of the compounds of theinvention. Uses of the compounds of the current invention include:killing or inhibiting the growth or replication of a tumor cell orcancer cell, treating cancer, treating a pre-cancerous condition,preventing the multiplication of a tumor cell or cancer cell, preventingcancer, preventing the multiplication of a cell that expresses anauto-immune antibody. These uses comprise administering to an animalsuch as a mammal or a human in need thereof an effective amount of acompound of the present invention.

The compound of the current invention is useful for treating diseasessuch as cancer in an animal, such as a human being. Compositions anduses for treating tumors by providing a subject the composition in apharmaceutically acceptable manner, with a pharmaceutically effectiveamount of a composition of the present invention are provided.

By “cancer” herein is meant the pathological condition in humans that ischaracterized by unregulated cell proliferation. Examples include butare not limited to: carcinoma, lymphoma, blastoma, and leukemia. Moreparticular examples of cancers include but are not limited to: lung(small cell and non-small cell), breast, prostate, carcinoid, bladder,gastric, pancreatic, liver (hepatocellular), hepatoblastoma, colorectal,head and neck squamous cell carcinoma, esophageal, ovarian, cervical,endometrial, mesothelioma, melanoma, sarcoma, osteosarcoma, liposarcoma,thyroid, desmoids, chronic myelocytic leukemia (AML), and chronicmyelocytic leukemia (CML).

By “inhibiting” or “treating” or “treatment” herein is meant toreduction, therapeutic treatment and prophylactic or preventativetreatment, wherein the objective is to reduce or prevent the aimedpathologic disorder or condition. In one example, followingadministering of a compound of the present invention, a cancer patientmay experience a reduction in tumor size. “Treatment” or “treating”includes (1) inhibiting a disease in a subject experiencing ordisplaying the pathology or symptoms of the disease, (2) ameliorating adisease in a subject that is experiencing or displaying the pathology orsymptoms of the disease, and/or (3) affecting any measurable decrease ina disease in a subject or patient that is experiencing or displaying thepathology or symptoms of the disease. To the extent a compound of thepresent invention may prevent growth and/or kill cancer cells, it may becytostatic and/or cytotoxic.

By “therapeutically effective amount” herein is meant an amount of acompound provided herein effective to “treat” a disorder in a subject ormammal. In the case of cancer, the therapeutically effective amount ofthe drug may either reduce the number of cancer cells, reduce the tumorsize, inhibit cancer cell infiltration into peripheral organs, inhibittumor metastasis, inhibit tumor growth to certain extent, and/or relieveone or more of the symptoms associated with the cancer to some extent.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order. As used herein, the term “pharmaceutical combination”refers to a product obtained from mixing or combining activeingredients, and includes both fixed and non-fixed combinations of theactive ingredients. The term “fixed combination” means that the activeingredients, e.g. a compound of Formula (1) and a co-agent, are bothadministered to a patient simultaneously in the form of a single entityor dosage. The term “non-fixed combination” means that the activeingredients, e.g. a compound of Formula (1) and a co-agent, are bothadministered to a patient as separate entities either simultaneously,concurrently or sequentially with no specific time limits, wherein suchadministration provides therapeutically effective levels of the activeingredients in the body of the patient. The latter also applies tococktail therapy, e.g. the administration of three or more activeingredients.

In some embodiments, the diseases condition is tumor.

In some embodiments, the disease condition comprises abnormal cellproliferation, such as a pre-cancerous lesion.

The current invention is particularly useful for the treatment of cancerand for the inhibition of the multiplication of a tumor cell or cancercell in an animal. Cancer, or a precancerous condition, includes atumor, metastasis, or any disease or disorder characterized byuncontrolled cell growth, can be treated or prevented by administrationthe drug-ligand complex of the current invention. The compound deliversthe activating moiety to a tumor cell or cancer cell. In someembodiments, the targeting moiety specifically binds to or associateswith a cancer-cell or a tumor-cell-associated antigen. Because of itsclose proximity to the ligand, after being internalized, the activatingmoiety can be taken up inside a tumor cell or cancer cell through, forexample, receptor-mediated endocytosis. The antigen can be attached to atumor cell or cancer cell or can be an extracellular matrix proteinassociated with the tumor cell or cancer cell. Once inside the cell, thelinker is hydrolytically or enzymatically cleaved by a tumor-cell orcancer-cell-associated proteases, thereby releasing the activatingmoiety. The released activating moiety is then free to diffuse andinduce or enhance immune activity of immune cells or tumor cells. In analternative embodiment, the activating moiety is cleaved from thecompound tumor microenvironment, and the drug subsequently penetratesthe cell.

Representative examples of precancerous conditions that may be targetedby the compounds of the present invention, include: metaplasia,hyperplysia, dysplasia, colorectal polyps, actinic ketatosis, actiniccheilitis, human papillomaviruses, leukoplakia, lychen planus andBowen's disease.

Representative examples of cancers or tumors that may be targeted bycompounds of the present invention include: lung cancer, colon cancer,prostate cancer, lymphoma, melanoma, breast cancer, ovarian cancer,testicular cancer, CNS cancer, renal cancer, kidney cancer, pancreaticcancer, stomach cancer, oral cancer, nasal cancer, cervical cancer andleukemia. It will be readily apparent to the ordinarily skilled artisanthat the particular targeting moiety used in the compound can be chosensuch that it targets the activating moiety to the tumor tissue to betreated with the drug (i.e., a targeting agent specific for atumor-specific antigen is chosen). Examples of such targeting moiety arewell known in the art, examples of which include anti-Her2 for treatmentof breast cancer, anti-CD20 for treatment of lymphoma, anti-PSMA fortreatment of prostate cancer and anti-CD30 for treatment of lymphomas,including non-Hodgkin's lymphoma.

In some embodiments, the abnormal proliferation is of cancer cells.

In some embodiments, the cancer is selected from the group consistingof: breast cancer, colorectal cancer, diffuse large B-cell lymphoma,endometrial cancer, follicular lymphoma, gastric cancer, glioblastoma,head and neck cancer, hepatocellular cancer, lung cancer, melanoma,multiple myeloma, ovarian cancer, pancreatic cancer, prostate cancer,and renal cell carcinoma.

In some embodiments, the present invention provides a compound for usein killing a cell. The compound is administered to the cell in an amountsufficient to kill said cell. In an exemplary embodiment, the compoundis administered to a subject bearing the cell. In a further exemplaryembodiment, the administration serves to retard or stop the growth of atumor that includes the cell (e.g., the cell can be a tumor cell). Forthe administration to retard the growth, the rate of growth of the cellshould be at least 10% less than the rate of growth beforeadministration. Preferably, the rate of growth will be retarded at least20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or completely stopped.

Effective Dosages

Pharmaceutical compositions suitable for use with the present inventioninclude compositions wherein the active ingredient is contained in atherapeutically effective amount, i.e., in an amount effective toachieve its intended purpose. The actual amount effective for aparticular application will depend, inter alia, on the condition beingtreated. Determination of an effective amount is well within thecapabilities of those skilled in the art, especially in light of thedetailed disclosure herein.

For any compound described herein, the therapeutically effective amountcan be initially determined from cell culture assays. Target plasmaconcentrations will be those concentrations of active compound(s) thatare capable of inhibition cell growth or division. In preferredembodiments, the cellular activity is at least 25% inhibited. Targetplasma concentrations of active compound(s) that are capable of inducingat least about 30%, 50%, 75%, or even 90% or higher inhibition ofcellular activity are presently preferred. The percentage of inhibitionof cellular activity in the patient can be monitored to assess theappropriateness of the plasma drug concentration achieved, and thedosage can be adjusted upwards or downwards to achieve the desiredpercentage of inhibition.

As is well known in the art, therapeutically effective amounts for usein humans can also be determined from animal models. For example, a dosefor humans can be formulated to achieve a circulating concentration thathas been found to be effective in animals. The dosage in humans can beadjusted by monitoring cellular inhibition and adjusting the dosageupwards or downwards, as described above.

A therapeutically effective dose can also be determined from human datafor compounds which are known to exhibit similar pharmacologicalactivities. The applied dose can be adjusted based on the relativebioavailability and potency of the administered compound as comparedwith the known compound.

Adjusting the dose to achieve maximal efficacy in humans based on themethods described above and other methods as are well-known in the artis well within the capabilities of the ordinarily skilled artisan.

In the case of local administration, the systemic circulatingconcentration of administered compound will not be of particularimportance. In such instances, the compound is administered so as toachieve a concentration at the local area effective to achieve theintended result.

For use in the prophylaxis and/or treatment of diseases related toabnormal cellular proliferation, a circulating concentration ofadministered compound of about 0.001 μM to 20 μM is preferred, withabout 0.01 μM to 5 μM being preferred.

Patient doses for oral administration of the compounds described herein,typically range from about 1 mg/day to about 10,000 mg/day, moretypically from about 10 mg/day to about 1,000 mg/day, and most typicallyfrom about 50 mg/day to about 500 mg/day. Stated in terms of patientbody weight, typical dosages range from about 0.01 to about 150mg/kg/day, more typically from about 0.1 to about 15 mg/kg/day, and mosttypically from about 1 to about 10 mg/kg/day, for example 5 mg/kg/day or3 mg/kg/day.

In at least some embodiments, patient doses that retard or inhibit tumorgrowth can be 1 μmol/kg/day or less. For example, the patient doses canbe 0.9, 0.6, 0.5, 0.45, 0.3, 0.2, 0.15, or 0.1 μmol/kg/day or less(referring to moles of the drug). Preferably, the antibody with drugconjugates retards growth of the tumor when administered in the dailydosage amount over a period of at least five days.

For other modes of administration, dosage amount and interval can beadjusted individually to provide plasma levels of the administeredcompound effective for the particular clinical indication being treated.For example, in one embodiment, a compound according to the inventioncan be administered in relatively high concentrations multiple times perday. Alternatively, it may be more desirable to administer a compound ofthe invention at minimal effective concentrations and to use a lessfrequent administration regimen. This will provide a therapeutic regimenthat is commensurate with the severity of the individual's disease.

Utilizing the teachings provided herein, an effective therapeutictreatment regimen can be planned which does not cause substantialtoxicity and yet is entirely effective to treat the clinical symptomsdemonstrated by the particular patient. This planning should involve thecareful choice of active compound by considering factors such ascompound potency, relative bioavailability, patient body weight,presence and severity of adverse side effects, preferred mode ofadministration and the toxicity profile of the selected agent.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

EXAMPLES

The present invention is further exemplified, but not limited, by thefollowing and Examples that illustrate the preparation of the compoundsof the invention.

Example 1 Generation of her2 or egfr Transfected L Cell Lines

Reagents: L cell was from ATCC (Manassas, Va.; Cat No CRL2648),Her2/pEZ-Lv105 or egfr/pCMV cDNA was purchased from Sino BiologicalInc., (Cat No H10004) or GeneCopoeia, (Cat No Z2866), Glucose DMEM,L-glutamine, Lipofectamine 2000 (Invitrogen; Carlsbad, Calif.).

To make cell lines for screening the conjugated Trastuzumab, L cellexpressing the tagged Her2 or EGFR were generated. The Her2/pEZ-Lv105 oregfr/pCMV cDNA construct was transfected into L cells (that grown inhigh glucose DMEM+10% FBS+2 mM L-glutamine cells) by the standardLipofectamine 2000 protocol.

Binding Analysis (FACS Analysis)

To determine binding capability of conjugated Trastuzumab, FACS analysisof L cells expressing human her2 were performance. Briefly,approximately 10⁶ cells of L cells with transient transfected her2 in100 μl were incubated with varying amounts of Trastuzumab conjugatedantibody, PBS or secondary antibody alone or irrelevant huIgG were usedas a negative control. After washing, cells were re-suspended in FACSbuffer and incubated 30 min at room temperature with 20 μL mouseanti-human IgG conjugated to phycoerythrin (Mu anti-Human-PE) secondaryantibody in a 100 μL reaction volume. After washing, cells were fixed in200 μL of 2% paraformaldehyde/PBS and flow cytometery performed. Thesame procedure was used for irrelevant human IgG antibody as an isotypecontrol to set baseline PMT per cell line. Flow cytomery was performedon a BD FACSCalibur® and geometric mean fluorescence intensity wasrecorded for each sample. Recorded data was analyzed using FlowJosoftware. Results are shown in FIG. 1.

Generation of Antibody with Toll Like Receptor Ligand Conjugates

Reagents: Trastuzumab (Roche/Genentech Corporation, South San Francisco,Calif.), Cetuximab (Merck,); MC-val-cit-PAB linked with resiquimod(MC-vc-PAB-TLRL) or MC-linked with resiquimod (MC-TLRL) (Synthesized inContract Research Organization, China), Sodium borate, Sodium chloride,Dithiothreitol (DTT), Sephadex G25, DTPA, DTNB, andMaleimidocaproyl-monomethyl (Sigma-Aldrich, Milwaukee, Wis.).

The drugs used for generation of antibody TLR ligand conjugates includedTrastuzumab and resiquimod (TLRL), in some case, Cetuximab was used forconjugates. The linkers used for generation of the TLAC were cleavablelinker MC-vc-PAB or non cleavable linker MC.

Preparation of Trastuzumab MC-TLRL or Cetuximab MC-TLRL

Trastuzumab was purified from HERCEPTIN® by buffer-exchange at 20 mg/mL,and antibody dissolved in 500 mM sodium borate and 500 mM sodiumchloride at pH 8.0 is treated with an excess of 100 mM dithiothreitol(DTT). After incubation at 37° C. for about 30 minutes, the buffer isexchanged by elution over Sephadex G25 resin and eluted with PBS with 1mM DTPA. The thiol/Ab value is checked by determining the reducedantibody concentration from the absorbance at 280 nm of the solution andthe thiol concentration by reaction with DTNB (Sigma-Aldrich, Milwaukee,Wis.) and determination of the absorbance at 412 nm. The reducedantibody dissolved in PBS is chilled on ice. The drug linker reagent,Mc-linked with resiquimod, dissolved in DMSO, is diluted in acetonitrileand water at known concentration, and added to the chilled reducedantibody in PBS. After about one hour, an excess of maleimide is addedto quench the reaction and cap any unreacted antibody thiol groups. Insome case, coupling to lysines of immunoglobulin with standard procedurewas performed. The reaction mixture is concentrated by centrifugalultrafiltration and conjugated antibody is purified and desalted byelution through G25 resin in PBS, filtered through 0.2 nm filters understerile conditions, and frozen for storage. Preparation of CetuximabMC-TLRL used the same procedure.

Preparation of Trastuzumab MC-vc-TLRL or Cetuximab MC-vc-TLRL

Antibodies were linked to TLRL through the cysteine byMaleimidocaproyl-valine-citruline (vc)-p-aminobenzyloxycarbonyl(MC-vc-PAB). The MC-vc-PAB linker is cleavable by intercellularproteases such as cathepsin B and when cleaved, releases free drug(Doronina et al., Nat. Biotechnol., 21: 778-784 (2003)) while the MClinker is resistant to cleavage by intracellular proteases. PurifiedTrastuzumab was dissolved in 500 mM sodium borate and 500 mM sodiumchloride at pH 8.0 and further treated with an excess of 100 MMdithiothreitol (DTT). After incubation at 37° C. for about 30 minutes,the buffer is exchanged by elution over Sephadex G25 resin and elutedwith PBS with 1 mM DTPA. The thiol/Ab value was checked by determiningthe reduced antibody concentration from the absorbance at 280 nm of thesolution and the thiol concentration by reaction with DTNB(Sigma-Aldrich, Milwaukee, Wis.) and determination of the absorbance at412 nm (extinction coefficient=13600 cm⁻¹ M⁻¹). The reduced antibodydissolved in PBS was chilled on ice. The MC-val-cit-PAB-PNP linked withresiquimod in DMSO, was dissolved in acetonitrile and water, and addedto the chilled reduced antibody in PBS. After one hour incubation, anexcess of maleimide was added to quench the reaction and cap anyunreacted antibody thiol groups. In some case, coupling to lysines ofimmunoglobulin with standard procedure was performed. The reactionmixture was concentrated by centrifugal ultrafiltration and the antibodydrug conjugate, was purified and desalted by elution through G25 resinin PBS, filtered through 0.2 μm filters under sterile conditions, andfrozen for storage. Preparation of Cetuximab MC-vc-TLRL was the sameprocedure.

Typically a conjugation reaction of antibody with MC-TLRL or MC-vc-TLRLresults in a heterogeneous mixture comprising antibodies differentnumbers of attached, conjugated TLRL drugs, i.e. drug loading where drugis a distribution from 1 to about 8. Thus, antibody MC-TLRL, or antibodyMC-vc-TLRL, includes isolated, purified species molecules as well asmixtures of average drug loading from 1 to 8. By control process, drugloads were in the range of 3-5. The average number of TLRL drug moietiesper antibody antibody in compound preparations from conjugationreactions may be characterized by conventional means such as massspectroscopy, ELISA assay, electrophoresis, and HPLC. The quantitativedistribution of antibody MC-TLRL or antibody MC-vc-TLRL in terms of drugmay also be determined By ELISA, the averaged value of drug payloadnumber in a particular preparation of antibody with TLRL conjugation maybe determined (Hamblett et al (2004) Clinical Cancer Res. 10:7063-7070;Sanderson et al (2005) Clinical Cancer Res. 11:843-852). However, thedistribution of drug values is not discernible by the antibody-antigenbinding and detection limitation of ELISA. Also, ELISA assay fordetection of antibody-drug conjugates does not determine where the drugmoieties are attached to the antibody, such as the heavy chain or lightchain fragments, or the particular amino acid residues. In someinstances, separation, purification, and characterization of homogeneousTrastuzumab MC-TLRL or Trastuzumab MC-vc-TLRL where drug is a certainvalue from Trastuzumab MC-TLRL or Trastuzumab MC-vc-TLRL with other drugloadings may be achieved by means such as reverse phase HPLC orelectrophoresis.

Antibody-Dependent Cell-Mediated Cytotoxicity Assay (ADCC)

Reagents: SKBR3 cells (ATCC, catalog #HTB-30), McCoy's 5A (Invitrogen,Cat No. 22400, Lot No. 747809); RPMI-1640 (Invitrogen, Cat No. 11835,Lot No. 764956); FCS (Hyclone, Cat No. SH30084.03, Lot No. GRH0054);Ficoll-Hypaque (Amersham Biosciences, Piscataway, N.J.; Cat No17-1440-02), 96 well plate (Costar, Cat No. 3599, Cat No. 3916); Trypanblue (Invitrogen Cat No 15250-061); LDH kit (Promega, Cat No. G7891),ELISA reader MD5 (Molecule device), Humanized antibody Herceptin®(Genentech Corporation, South San Francisco, Calif.; trade nameTrastuzumab) was reconstituted with water to make a 10 mg/ml stocksolution before use.

To determine if Trastuzumab conjugated with a high payload of TLRL wouldstill have effector function, antibody-dependent cell-mediatedcytotoxicity (ADCC) activities of the several different form ofconjugation were tested and compared with the ADCC activity ofTrastuzumab reference material which has been shown to havesignificantly enhanced ADCC activity. ADCC assays were carried out usingperipheral blood mononuclear cells (PBMCs) from healthy donors aseffector cells, and a human SKBR3 cell line as target cells. On firstday, 1×10⁴/100 uL of SKBR3 cells was seeded into the 96 well platesfollowed by incubation at 37° C. with 5% CO2 for 48 hours.

On third days, human PBMC was prepared fresh from human blood obtainedfrom healthy volunteer donors. Human venous blood samples were collectedin ammonium citrate (ACD-A) tubes. The tubes were inverted severaltimes, and whole blood was transferred into one 50 ml conical tube.Blood was diluted 1:3 with PBS in 2% FBS. Ficoll-Hypaque was dispensedslowly underneath blood/PBS mixture. Samples were centrifuged at roomtemperature at 2400 rpm for 30 minutes before removing the upper layer(plasma/PBS) by aspiration. Buffy coats were collected with sterilepipettes and pooled into a 50 ml conical tube. If visible clumps of WBCremained below the buffy coat, all of the WBC material was collected,with care not to remove excess Ficoll-Hypaque. Sterile PBS-2% FBS wasadded and mixed by inversion. The diluted PBMC suspensions wascentrifuged at 250×g at room temp for 20 minutes, and the cell pelletwas collected. The PBMC pellet was suspended in RPMI-1640 medium with 2%heat-inactivated FBS, washed and the number of viable cells was checkedby Trypan blue exclusion. 1.2×10⁷ cells/mL in density were prepared andready to be used. Meanwhile the final concentrations of antibodies theranged from 12, 4, 1.2, 0.4, 0.12, 0.04, 0.012, 0.004, to 0 μg/mL werewell prepared in RPMI-1640.

Serial dilutions of test and control antibodies then were added to wellscontaining the target cells, PBMC effector cells in 50 μL of RPMI-1640medium were added to each well with effector:target cell ratio of 60:1and incubated for an additional 17 hours. The plates were centrifuged atthe end of incubation and the supernatants were assayed for lactatedehydrogenase (LDH) activity using a LDH measuring kit. Cell lysis wasquantified through absorbance at 490 nm using a microplate reader. Theabsorbance of wells containing only the target cells served as thecontrol for background, whereas wells containing target cells lysed withTriton-X100 provided maximum signal available. Antibody-independentcellular cytotoxicity was measured in wells containing target andeffector cells without the addition of antibody. Cytotoxicity wascalculated according to the following equation:

% Cytotoxicity=100%×[A490 nm (Sample)−A490(target cell)−A490(effectcells]/[A490 nm (Target cells lysed)−A490 nm (target cells)]

The mean ADCC values from duplicates of sample dilutions were plottedagainst the antibody concentration, and the EC50 values and the maximumextent of ADCC (%) were generated by fitting into Prism 5 (GraphPad).

Enrichment of Human Dendritic Cells (DCs) from PBMC

Human PBMC was prepared from Buffy coats obtained from healthy volunteerdonors by Ficoll centrifugation. Dendritic cells were enriched by usingnegative depletion with magnetic beads (Miltenyi Biotec) with mixture ofanti-CD3, CD19, CD20, CD14, and CD16 antibodies from human PBMC. Theenrichment of DCs was stained with goat anti-mouse FITC (lineages),HLA-DR-APCCy7, CD123-BV421 and CD11C-APC. The stained cells wereanalyzed on BD LSR Fortessa. The anti-CD3, CD4, CD11C, CD19, CD14, CD16,CD123 monoclonal antibody were purchased from BD Biosciences orBiolgend.

FIG. 3( a) shows the percentages of DCs before and post enrichment. Thenumbers in upper two plots represent the percentages of DCs(HLA-DR+Lin−) of total cells before and after lineage depletion. Thenumbers in lower plots represent percentages of mDC (CD11C+CD123−) andpDC (CD123+CD11C−) of total DCs before and after lineage depletion.

Stimulation of Enriched Human DCs and Cytokines Expression

1-2×10⁵ enriched DCs were plated in a 96-well plate in 100 μl media, 100μl diluted stimulators were add to the plate and cultured for 20-22 h in37° C. incubator. The supernatant were collected and human IFN-α, IL-6,IL-12(p70) and TNF-α were analyzed by ELISA (Mabtech AB).

Statistical Analysis

The significance of all comparisons was calculated using a Student'stwo-tailed t test assuming unequal variance between mock and samplegroups, and results considered significant when p<0.05. Correlationsbetween parameters were assessed using Spearman's rank correlation test,P values<0.05 were consider to be statistically significant.

In Vivo Tumor Cell Killing Assay Using Trastuzumab TLRL Conjugates orCetuximab TLRL Conjugates

For development of patient-derived gastric carcinoma xenograft (PDX)mouse models, female Balb/c nude mice (from SLAC, Shanghai, China) of6-8 weeks old were used for tumor fragment implantation. Animals werefed with normal nude mice diet and housed in SPF animal facility inaccordance with the Guide for Care and Use of Laboratory Animals andregulations of the Institutional Animal Care and Use Committee. PatentSTO#69 gastric tumor fragments of about 15-30 mm³ in size were implanted(s.c.) into right flanks of Balb/c nude mice.

For development of lung cancer xenograft models, Female Balb/c nude miceof 6-8 weeks old were used for tumor fragment implantation. Human lungcancer cell line H1650 (ATCC, Cat. # CRL5883) were cultured in RPMI-1640medium containing 10% serum, and implanted (s.c.) into right flanks ofBalb/c nude mice. Each mouse received 2×10⁶ cells per inoculation in 100μl of matrigel.

Drugs were administered by i.v. route with 5˜20 mg/kg of antibody or 20mg/kg of Tarceva or reference drug, QWKx3. Tumors were measured once aweek by caliper to determine its subcutaneous growth. Tumors weremeasured twice a week in two dimensions with calipers. Tumor volume wascalculated using following formula: tumor volume=(length×width2)×0.5.Average tumor volumes or body weights were plotted using graph programPrism 5 (GraphPad). An endpoint for efficacy study was set at 30-45 dayspost-first treatment or when a tumor size reached above 2000 mm3,whichever came first. If a mouse lose more than 20% of body weight or isvery sick and cannot get to adequate food or water, it will be removedfrom the study and euthanized. Tumors were collected from mice at theend point, half frozen in LN₂ and half fixed in formalin for preparingFFPE tissues.

1. A compound having the structure of Formula (I): TM-Ln-AM  (I), wherein TM is a targeting moiety, AM is an activating moiety that is capable of activating a human dendritic cell, NK cell, macrophage, or tumor cell, or a combination thereof, Ln is a linker, and n is an integer selected from 0 and 1, with the proviso that when TM comprises an antibody, AM does not comprise a CpG oligonucleotide.
 2. The compound of claim 1, wherein said human dendritic cell is a plasmacytoid dendritic cell.
 3. The compound of claim 1, wherein said human dendritic cell is a myeloid dendritic cell.
 4. (canceled)
 5. The compound of claim 1, wherein said AM is capable of binding specifically to human TLR7 and TLR8.
 6. (canceled)
 7. (canceled)
 8. (canceled)
 9. The compound of claim 1, wherein said targeting moiety is capable of binding to a tumor cell specifically or preferably in comparison to a non-tumor cell.
 10. The compound of claim 9, wherein said the tumor cell is of a carcinoma, a sarcoma, a lymphoma, a myeloma, or a central nervous system cancer.
 11. The compound of claim 1, wherein said targeting moiety is capable of binding to a tumor antigen specifically or preferably in comparison to a non-tumor antigen.
 12. The compound of claim 11, wherein said tumor antigen is selected from the group consisting of: CD2, CD19, CD20, CD22, CD27, CD33, CD37, CD38, CD40, CD44, CD47, CD52, CD56, CD70, CD79, and CD137.
 13. The compound of claim 11, wherein said tumor antigen is selected from the group consisting of: 4-1BB, 5T4, AGS-5, AGS-16, Angiopoietin 2, B7.1, B7.2, B7DC, B7H1, B7H2, B7H3, BT-062, BTLA, CAIX, Carcinoembryonic antigen, CTLA4, Cripto, ED-B, ErbB1, ErbB2, ErbB3, ErbB4, EGFL7, EpCAM, EphA2, EphA3, EphB2, FAP, Fibronectin, Folate Receptor, Ganglioside GM3, GD2, glucocorticoid-induced tumor necrosis factor receptor (GITR), gp100, gpA33, GPNMB, ICOS, IGF1R, Integrin an, Integrin anb, KIR, LAG-3, Lewis Y, Mesothelin, c-MET, MN Carbonic anhydrase IX, MUC1, MUC16, Nectin-4, NKGD2, NOTCH, OX40, OX40L, PD-1, PDL, PSCA, PSMA, RANKL, ROR1, ROR2, SLC44A4, Syndecan-1, TACI, TAG-72, Tenascin, TIM3, TRAILR1, TRAILR2, VEGFR-1, VEGFR-2, VEGFR-3, and variants thereof.
 14. The compound of claim 1, wherein said targeting moiety comprises an immunoglobulin, a protein, a peptide, a small molecule, a nanoparticle, or a nucleic acid.
 15. The compound of claim 14, wherein said targeting moiety comprises an antibody, or a functional fragment thereof.
 16. The compound of claim 15, the antibody is selected from the group consisting of: Rituxan (rituximab), Herceptin (trastuzumab), Erbitux (cetuximab), Vectibix (Panitumumab), Arzerra (Ofatumumab), Benlysta (belimumab), Yervoy (ipilimumab), Perjeta (Pertuzumab), Tremelimumab, Nivolumab, Dacetuzumab, Urelumab, MPDL3280A, Lambrolizumab, and Blinatumomab.
 17. The compound of claim 15, wherein said targeting moiety comprises a Fab, Fab′, F(ab′)2, single domain antibody, T and Abs dimer, Fv, scFv, dsFv, ds-scFv, Fd, linear antibody, minibody, diabody, bispecific antibody fragment, bibody, tribody, sc-diabody, kappa (lamda) body, BiTE, DVD-Ig, SIP, SMIP, DART, or an antibody analogue comprising one or more CDRs.
 18. The compound of claim 14, wherein said targeting moiety comprises a ATWLPPR polypeptide of VEGFR, Thrombospondin-1 mimetics, CDCRGDCFCG (cyclic) polypeptide, SCH 221153 fragment, NCNGRC (cyclic) polypeptide, CTTHWGFTLC polypeptide, CGNKRTRGC polypeptide (LyP-1), Octreotide, Vapreotide, Lanreotide, C-3940 polypeptide, Decapeptyl, Lupron, Zoladex, or Cetrorelix.
 19. The compound of claim 14, wherein said targeting moiety comprises folic acid or a derivative thereof.
 20. The compound of claim 14, wherein said targeting moiety comprises extracellular domains (ECD) or soluble form of PD-1, CTLA4, BTLA, KIR, TIM3, 4-1 BB, LAG3, full length of partial of a surface ligand amphiregulin, betacellulin, EGF, ephrin, epigen, epiregulin, IGF, neuregulin, TGF, TRAIL, or VEGF.
 21. (canceled)
 22. The compound of claim 14, wherein said targeting moiety comprises an aptamer.
 23. (canceled)
 24. (canceled)
 25. The compound of claim 1, wherein said linker is selected from the group consisting of a hydrazine group, a polypeptide, a disulfide group, and a thioether group.
 26. The compound of claim 1, wherein said linker is enzymatic cleavable.
 27. The compound of claim 1, wherein said linker is not enzymatic cleavable.
 28. A pharmaceutical composition comprising a compound as claimed in claim 1, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers.
 29. The pharmaceutical composition of claim 28, further comprising a second chemotherapeutic agent.
 30. The pharmaceutical composition of claim 29, wherein said chemotherapeutic agent is selected from the group consisting of tamoxifen, raloxifene, anastrozole, exemestane, letrozole, imatanib, paclitaxel, cyclophosphamide, lovastatin, minosine, gemcitabine, cytarabine, 5-fluorouracil, methotrexate, docetaxel, goserelin, vincristine, vinblastine, nocodazole, teniposide etoposide, gemcitabine, epothilone, vinorelbine, camptothecin, daunorubicin, actinomycin D, mitoxantrone, acridine, doxorubicin, epirubicin, or idarubicin.
 31. A method for treating a disease condition in a subject that is in need of such treatment, comprising: administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of the compound of claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutical acceptable carrier.
 32. The method of claim 31, wherein said diseases condition is tumor.
 33. The method of claim 31, wherein said disease condition comprises abnormal cell proliferation.
 34. The method of claim 33, wherein said abnormal cell proliferation comprises a pre-cancerous lesion.
 35. The method of claim 33, wherein, wherein said abnormal proliferation is of cancer cells.
 36. The method of claim 35, wherein said cancer is selected from the group consisting of: breast cancer, colorectal cancer, diffuse large B-cell lymphoma, endometrial cancer, follicular lymphoma, gastric cancer, glioblastoma, head and neck cancer, hepatocellular cancer, lung cancer, melanoma, multiple myeloma, ovarian cancer, pancreatic cancer, prostate cancer, and renal cell carcinoma.
 37. (canceled)
 38. The compound of claim 1, wherein said activating moiety comprises a TLR ligand (TLRL), a Nod-Like Receptor ligand, a RIG-Like Receptor ligand, a CLR ligand, a CDS ligand, or a inflammasome inducers.
 39. The compound of claim 38, wherein said activating moiety comprises a ligand of one or more TLRs selected from the group consisting of: TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, TLR7/TLR8, TLR9, and TLR10.
 40. The compound of claim 39, wherein said activating moiety comprises: (i) a ligand of TLR2 selected from the group consisting of: (a) a heat killed bacteria product, preferably HKAL, HKEB, HKHP, HKLM, HKLP, HKLR, HKMF, HKPA, HKPG, or HKSA, HKSP, and (b) a cell-wall components product, preferably LAM, LM, LPS, LTA, LTA, PGN, FSL, Pam2CSK4, Pam3CSK4, or Zymosan; (ii) a ligand of TLR3 selected from the group consisting of: Poly (I:C) and Poly (A:U); (iii) a ligand of TLR4 selected from the group consisting of: LPS and MPLA; (iv) a ligand of TLR5 selected from the group consisting of: FLA and Flagellin; (v) a ligand of TLR7/8 selected from the group consisting of: ORN02, ORN06, ssPoly(U), ssRNA40, ssRNA41, ssRNA-DR, Poly(dT), CL075, CL097, CL264, CL307, Gardiquimod, Loxoribine, Imiquimod, and Resiquimod; (vi) a ligand of TLR9 selected from the group consisting of: ODN1585, ODN1668, ODN1826, ODN2006, ODN2007, ODN2216, ODN2336, ODN2395, and ODN M362; (vii) a ligand of TLR10; (viii) a ligand of nucleotide-oligomerization domain (NOD)-like selected from the group consisting of: NOD1 agonist (C12-iE-DAP, iE-DAP, Tri-DAP), NOD2 agonist (L18-MDP, MDP, M-TriLYS, M-TriLYS-D-ASN, Murabutide, N-Glycolyl-MDP), and NOD1/NOD2 agonists (M-TriDAP, PGN); (ix) a ligand of one or more RIG-I-Like receptors (RLR) selected from the group consisting of: 5′ppp-dsRNA, Poly (dA:dT), Poly(dG:dC), and Poly (I:C); (x) a ligand of one or more C-type lectin receptors (CLR) selected from the group consisting of: Curdlan AL, HKCA, HKSC, WGP, Zymosan, and Trehalose-6,6-dibehenate; (xi) a ligand of one or more Cytosolic DNA Sensors (CDS) selected from the group consisting of: c-GMP, HSV-60, ISD, pCpG, Poly (dA:dT), Poly(dG:dC), Poly (dA), and VACV-70; or (xii) an inflammasome inducer selected from the group consisting of: (a) NLRP3 inflammasome protein complex, preferably alum Crystals, ATP, CPPD Crystals, Hermozoin, MSU Crystals, Nano-SiO2, Nigericin, and (b) AIM2 inflammasome protein complex, preferably Poly (dA:dT).
 41. The compound of claim 37, wherein said activating moiety comprises IMO2134, IMO205, MGN-1703, MGN-1704, Agatolimod, SD-101, QAX-935, DIMS0150, Pollinex Quattro, OM-174, Eritoran, TAK-242, NI-0101, Type I interferon, type II interferon, type III interferon, IL-12, IL-23, IL-18, IL-7 or IL-15. 